BIOLOGY 
R 
6 


A  MANUAL 


OF 


BACTERIOLOGY 


BY 


HERBERT  U.  WILLIAMS,  M.  D. 

>\ 

Professor  of  Pathology  and  Bacteriology,  Medical  Department, 
University  of  Buffalo 


REVISED  BY 

B.  MEADE  ^OLTON,  M.  D. 

WASHINGTON,  D.  C. 

One  Time  Associate  in  Bacteriology,  Johns  Hopkins  University;  Chief  of  the  Bureau  of 

Health  Laboratory,  Philadelphia,  Penna.;  Professor  of  Pathology  and  Bacteriology, 

University  of  Missouri;  Bacteriologist,  Bureau  of  Animal  Industry,  etc. 


WITH  113  ILLUSTRATIONS 


FIFTH  EDITION,  REVISED  AND  ENLARGED 


Of    THE 

UNIVERSITY 

PHILADELPHIA 

:ofi*  & 


1012    WALNUT    STREET 
1908 


DIOLOGY 
R 
G 


Copyright,  1908 
By  HERBERT  U.  WILLIAMS. 


Printed  by 

The  Maple  Press 

York,  Pa. 


i 

•) 


PREFACE  TO  THE  FIFTH  EDITION. 


IN  the  preface  to  the  second  edition  of  this  manual  the 
author  states  that:  "the  purpose  of  this  book  is  to  give  in 
the  smallest  possible  space  the  facts  which  a  physician  must 
know,  with  some  of  those  which  it  is  desirable  that  he  should 
know,  and  a  little  of  that  which  he  may  learn  if  his  needs  or 
inclinations  lead  him  to  go  further."  While  n  the  present 
edition  this  purpose  has  been  kept  in  mind  by  the  reviser, 
nevertheless  it  has  been  necessary  to  make  many  alterations 
in  order  to  place  the  work  abreast  of  the  progress  which  has 
been  made  since  the  publication  of  the  last  edition.  Some 
things  which  have  become  obsolete  have  been  omitted,  much 
that  is  new  has  been  added,  and  the  scope  has  been  somewhat 
widened. 

Since  the  book  is  designed  primarily  for  medical  students, 
the  method  of  expression,  as  in  former  editions,  is  often  more 
definite  than  is  perhaps  justifiable,  and  it  must  be  understood 
that  this  form  is  adopted  merely  in  order  to  make  a  clear 
presentation  of  the  subject,  not  that  the  matter  is  in  every 
such  case  finally  settled,  nor  that  the  views  as  stated  are 
necessarily  shared  by  the  author  or  the  reviser,  but  that  they 
are  held  by  those  whose  opinions  are  worthy  of  consideration, 
and  are  the  interpretation  of  the  results  of  painstaking  investi- 
gation, but  which,  nevertheless,  may  be  in  time  modified  or 
abandoned  as  a  result  of  further  research. 

Those  who  are  familiar  with  the  former  editions  of  the 
book  will  find  that  the  arrangement  of  chapters  is  the  same  as 
heretofore.  Many  references  to  the  literature  have  been  added, 

v 

on/?./*  /I  I 


Vi  PREFACE. 

but  these  are  by  no  means  exhaustive,  many  had  to  be  omitted 
with  regret.  The  hygienic  examination  of  milk  has  been 
practically  rewritten;  the  hygienic  examination  of  water  con- 
siderably expanded.  The  chapter  on  disinfectants,  and  that 
on  surgical  antisepsis  have  been  also  greatly  altered.  The 
interesting  theoretical  subject  of  the  diversion  of  complement 
has  been  added  to  the  chapter  on  immunity.  The  trypano- 
somes  and  the  amoeba  have  been  given  additional  space,  but 
certainly  not  more  than  these  increasingly  important  micro- 
organisms demand.  The  index  has  been  not  only  greatly 
enlarged,  but  the  references  have  been  made  much  fuller  and 
more  specific. 

A  very  full  card  catalogue  of  references  prepared  by 
Dr.  Williams  was  made  use  of  in  looking  up  most  of  the 
articles  which  have  been  cited. 

As  in  the  fourth  edition,  the  author  is  relieved  of  all 
responsibility  for  the  statements  made  in  the  present  edition; 
both  for  those  retained  from  former  editions,  as  well  as  for 
those  which  have  been  added  by  the  reviser. 

B.  MEADE  BOLTON. 

WASHINGTON,  D.  C., 


PREFACE  TO  THE  THIRD  EDITION. 


The  plan  used  in  the  preceding  editions  of  this  manual  has 
been  followed  in  the  preparation  of  the  present  one.  The 
only  departures  have  been  in  the  insertion  of  a  short  histor- 
ical sketch  and  the  freer  use  of  references  to  original  articles 
and  reviews.  It  is  hoped  that  these  features  will  assist  in 
arousing  the  interest  of  students.  As  far  as  possible,  refer- 
ence has  been  made  tox  articles  in  American  and  English 
journals  likely  to  be  easy  of  access.  Besides  the  ones  just 
named,  numerous  additions  have  been  made  which  the  recent 
advances  in  our  knowledge  have  rendered  necessary.  Most 
of  the  illustrations  of  apparatus  are  new.  The  photomicro- 
graphs also  are  new  and  original,  with  a  few  exceptions  noted 
in  the  text.  It  is  probably  needless  to  say  that  none  of  them 
were  retouched.  The  writer  is  indebted  to  the  Gratwick 
Laboratory,  of  Buffalo,  for  the  use  of  its  facilities  in  making 
these  photographs.  H.  U.  W. 

BUFFALO,  NEW  YORK,  August,  1903. 


VI 1 


CONTENTS. 


PAGE 

Introduction  with  Historical  Sketch  .  i 


PART   I. 
BACTERIOLOGICAL  TECHNIC. 

CHAPTER  I. 

Examination   of   Bacteria  with  the   Microscope,   Including    Methods   of 

Staining 18 

CHAPTER  II. 
Sterilization 53 

CHAPTER  III. 
Culture-media 64 

CHAPTER  IV. 

The  Cultivation  of  Bacteria.     Tube-cultures;  the  Incubator;  Anaerobic 

Methods 78 

CHAPTER  V. 

The  Cultivation  of  Bacteria  (Continued).     Isolation  of  Bacteria;  Plate- 
cultures  94 

CHAPTER  VI. 
Inoculation  of  Animals.     Autopsies;  Collodion  Sacs 102 

CHAPTER  VII. 
Collection  of   Material 107 

CHAPTER  VIII. 

Systematic  Study  of   Species  of  Bacteria.     Suggestions  for  Class-work; 

Rules 112 

ix 


X  CONTENTS. 

PART  II. 

EXPERIMENTAL  STUDY  AND  PRACTICAL  APPLICATIONS. 
CHAPTER  I. 

PAGE 

Classification;  General  Morphology  and  Physiology  of  Bacteria   ....    117 

CHAPTER  II. 
Products  of  the  Growth  of  Bacteria 128 

CHAPTER    III. 
Bacteria  of  Soil,  Air,  Water  and  of  Milk  and  Other  Foods 135 

CHAPTER  IV. 
Bacteria  of  the  Normal  Human  Body .    160 

CHAPTER  V. 
Bacteria  in  Disease 167 

CHAPTER  VI. 

Bacterial  Poisons;  Agglutinins;  Lysins;  Precipitins 186 

CHAPTER  VII. 

Immunity;    Phagocytosis;   Antitoxin 198 

CHAPTER  VIII. 

Disinfection,  Sterilization  and  Antisepsis 238 

CHAPTER   IX. 
Surgical  Antisepsis 262 

PART  III. 
NON-PATHOGENIC  BACTERIA 267 

PART  IV. 

PATHOGENIC  BACTERIA 282 

PATHOGENIC  PROTOZOA 407 

APPENDIX 426 

INDEX 438 


LIST  OF  ILLUSTRATIONS. 


FIG.  PAGE 

1.  Micrococci.  Bacilli,  Spirilla 4 

2.  Test-tube  with  Culture-medium 7 

3.  Microscope ig 

4.  Abbe  Condenser 20 

5.  Platinum  Wires 22 

6.  Hanging-drop 23 

7.  Cornet  Forceps  for  Cover-glasses 26 

8.  Stewart  Forceps  for  Cover-glasses 26 

9.  Kirkbride  Forceps  for  Slides 27 

10.  Schanze  Microtome 39 

11.  Hot-air  Sterilizer 54 

12.  Arnold  Steam  Sterilizer 57 

13.  Massachusetts  Board  of  Health  Sterilizer 53 

14.  Koch  Steam  Sterilizer 59 

15.  Autoclave 61 

16.  Kitasato  Filter 62 

17.  Test-tube  with  Potato    .    .    . 72 

18.  Wire  Basket  for  Test-tubes 77 

19.  Manner  of  Holding  Test-tubes 79 

20.  Stab-culture 80 

21.  Smear-culture 80 

22.  Incubator 83 

23.  Reichert  Gas-regulator 84 

24.  Gas-regulator,  mercurial 84 

25.  Roux  Bimetallic  Gas-regulator 85 

26.  Koch  Automatic  Gas-burner 86 

27.  Rogers'  Electric  Thermo -regulator 88 

28.  Buchner's  Method  for  Cultivating  Anaerobes 90 

29.  Frankel's  Method  for  Cultivating  Anaerobes 91 

30.  Novy's  Method  for  Cultivating  Anaerobes 92 

31.  Streak  Culture  of  the  Potato  Bacillus 93 

32.  Petri  Dish 96 

33.  Dilution-cultures  in  Esmarch  Roll-tubes 97 

34.  Appearance  of  Colonies  on  Gelatin  in  a  Petri  Dish 98 

xi 


xii  LIST    OF    ILLUSTRATIONS. 

FIG.  PAGE 

35.  Esmarch's  Roll-tube 99 

36.  Mouse-holder i°2 

37.  Apparatus  for  the  Subcutaneous  Insertion  of  Solid  Substances  ...  103 

38.  McCrae's  Method  for  Making  Collodion  Capsules 106 

39.  Cover-glass  Preparation  of  Blood 108 

40.  Sternberg  Bulb •    •  109 

41.  Micrococci  of  Various  Forms 118 

42.  Bacilli  of  Various  Forms 119 

43.  Spirilla  of  Various  Forms 119 

44.  Involution  Forms 120 

45.  Bacteria  with  Capsules 122 

46.  Bacteria  with  Spores 123 

47.  Bacteria  Showing  Flagella 124 

48.  Fermentation-tube 132 

49.  Sedg wick- Tucker  Aerobioscope 138 

50.  Receptors  of  the  First  Order  Uniting  with  Toxin 219 

51.  Receptors  of  the  Second  Order  and  of  Some  Substance  Uniting  with 

One  of  Them 220 

52.  Receptors  of  the  Third  Order  and  of  Some  Substance  Uniting  with 

One  of  Them 222 

53.  "Spectrum"  of  Theoretically  Fresh  Crude  Toxin 224 

54.  "  Spectrum  "  of  Very  Fresh  Crude  Toxin 225 

55.  "  Spectrum-"  of  Crude  Toxin  as  it  is  Supposed  Always  Practically  to 

Occur 225 

56.  Mechanism  of  Bacteriolysis 230 

57.  Diversion  of  Complement  in  Undiluted  Immune  Serum 233 

58.  Partial  Bacteriolysis,  etc 234 

59.  Bacillus  subtilis 272 

60.  Spirilla  from  Swamp  Water 275 

61.  Spirilla  from  Swamp  Water  with  Flagella 276 

62.  Yeast  Cells 278 

63.  Penicillium  glaucum,    Oidium   lactis,   Aspergillus  glaucus,   Mucor 

mucedo 279 

64.  Staphylococcus  pyogenes  aureus  in  Pus 289 

65.  Staphylococcus  pyogenes  aureus  in  Pure  Culture 291 

66.  Staphylococcus  pyogenes  aureus  in  Culture  in  Gelatin 299 

67.  Streptococcus  pyogenes,  Pure  Culture 296 

68.  Streptococcus  pyogenes  in  Pus 297 

69.  Streptococcus  pyogenes  Culture  on  Agar 298 

70.  Micrococcus  tetragenus  in  Pus 299 

"71.  Micrococcus  lanceolatus  (of  Pneumonia)  in  Sputum 304 

72.  Micrococcus  lanceolatus  (of  Pneumonia)  Showing  Capsules  ....  305 

73.  Diplococcus  intracellularis  meningitidis 309 


LIST    OF   ILLUSTRATIONS.  Xlll 

FIG.  PAGE 

74.  Gonococcus  in  Pus 312 

75.  Bacillus  pyocyaneus 318 

76.  Bacillus  of  Bubonic  Plague 320 

77.  Bacillus  aerogenes  capsulatus 324 

78.  Bacillus  aerogenes  capsulatus  Culture 326 

79.  Bacillus  of  Tetanus • 328 

80.  Bacillus  of  Anthrax 331 

ST.  Bacillus  of  Anthrax  with  Spores 332 

82.  Bacillus  of  Anthrax  Colony 333 

83.  Bacillus  of  Anthrax  Culture 334 

84.  Bacillus  of  Anthrax  Showing  Concave  Ends 335 

85.  Bacillus  of  Anthrax  in  the  Liver 336 

86.  Bacillus  of  Diphtheria 339 

87.  Bacillus  of  Diphtheria,  Neisser's  Stain 340 

88.  Tubes  for  Cultivation  of  Diphtheria  Bacillus 340 

89.  Bacillus  of  Diphtheria,  Culture 341 

90.  Bacillus  tuberculosis 350 

91.  Branching  Form  of  Tubercle  Bacillus •  .    .    .    .   351 

92.  Bacillus  tuberculosis,  stained,  in  Sputum 352 

93.  Ray -fungus  of  Actinomycosis,  Fresh  Preparation 367 

94.  Actinomyces  bovis  from  a  Pure  Culture 369 

95.  Bacillus  of  Typhoid  Fever 370 

96.  Bacillus  of  Typhoid  Fever  with  Flagella 371 

97.  Widal  Serum-reaction  with  Typhoid  Bacilli 376 

98.  Bacillus  coli  communis 382 

99.  Bacillus  coli  communis  with  Flagella 383 

100.  Spirillum  of  Cholera 388 

101.  Spirillum  of  Cholera  Involution  Forms 391 

102.  Spirillum  of  Cholera  Colonies  on  Gelatin  Plates 392 

103.  Spirillum  of  Cholera  Culture  in  Gelatin 393 

104.  Vibrio  proteus 4°i 

105.  Spirillum  of  Relapsing  Fever 4°3 

106.  Malarial  Parasite 417 

107.  Malarial  Parasite 417 

108.  Malarial  Parasite 4*7 

109.  Malarial  Parasite 4*7 

no.  Trypanosomes  in  the  Blood  of  the  Rat 421 

in.  Plates  for  Counting  Colonies 440,441,442 


INTRODUCTION. 


BACTERIOLOGY  is  not  a  subject  which  one  should  attempt  to 
learn  from  books  alone  or  without  instructors;  for  aside  from 
the  difficulty  or  impossibility  of  such  a  task,  there  may  be 
danger  as  well.  Indeed  the  warning  should  perhaps  be  given  in 
the  beginning  that  in  view  of  the  fact  that  so  many  bacteria  cause 
diseases  which  not  only  affect  the  individual  who  suffers,  but 
makes  him  a  menace  to  those  about  him,  no  one  is  justified  in 
entering  upon  the  study  without  proper  guidance. 

Anyone  who  has  not  himself  worked  in  a  bacteriological 
laboratory  finds  it  difficult  to  form  a  vivid  conception  of  what 
bacteria  are  like,  because  among  the  familiar  animals  and 
plants  there  are  none  with  which  a  close  comparison  can  be 
made.  Of  the  common  organisms,  perhaps  ordinary  yeasts 
and  moulds  are  most  like  the  bacteria.  Yeasts  and  moulds,  as 
everyone  knows,  grow  on  bread,  cheese,  meat,  syrups  and  the 
like.  They  flourish  in  moist  and  dark  places,  as  do  mush- 
rooms, puffballs  and  the  other  fungi.  All  these  fungi,  ap- 
pearing so  different  in  some  respects,  are  alike  in  one  par- 
ticular, which  is  the  absence  of  the  green  color  that  we  are  apt  to 
think  of  as  being  the  essential  feature  of  vegetation.  Plants 
that  are  green  owe  their  color  to  a  substance  called  chlorophyll. 
Upon  the  properties  of  this  substance  one  of  the  most  funda- 
mental vital  phenomena  in  biology  depends.  By  means  of 
chlorophyll,  under  the  influence  of  sunlight,  plants  are  able  to 
use  as  food  the  carbon  dioxide  which  is  always  present  in  the 
atmosphere  in  small  amounts.  Although  carbon  dioxide  is 
one  of  the  simplest  and  most  stable  of  compounds,  its  com- 
ponent elements  are  dissociated  by  the  plant,  and  employed  in 


2  MANUAL    OF    BACTERIOLOGY. 

the  formation  of  other  much  more  complex  and  unstable  com- 
pounds, such  as  starch  and  cellulose,  which  enter  into  the 
plant's  structure.  The  work  of  plants,  it  will  be  noticed,  is  in 
the  main,  precisely  the  reverse  of  that  performed  by  animals. 
Animals  take  the  unstable  carbohydrates  with  high  potential 
energy,  such  as  starches  and  sugars,  as  food,  and  exhale  the 
stable  carbon  dioxide  from  the  lungs.  At  the  same  time  the 
animal  receives  the  benefit  of  the  energy  resulting  from  the 
oxidation  of  the  carbohydrates,  which*  may  appear  indirectly  in 
the  form  of  nervous  or  muscular  activity  or  warmth. 

Those  plants  that  are  devoid  of  chlorophyll  are  compelled  to 
some  extent  to  use  the  same  kinds  of  food  as  animals.  They 
are  unable  to  decompose  carbon  dioxide  (in  most  cases),  and 
procure  their  nourishment  from  substances  derived  from  the 
dead  or  living  bodies  of  other  plants  or  animals.  Since  they 
have  no  chlorophyll,  light  is  of  no  advantage  to  them,  and  is 
often  a  positive  detriment.  Bacteria  contain  no  chlorophyll, 
and  consequently  are  unable  to  decompose  carbon  dioxide  and 
to  use  it  as  food.* 

There  is  another  well-known  property,  possessed  by  yeasts 
especially,  which  may  be  useful  in  explaining  the  work  done  by 
bacteria.  It  is  a  fact  of  every-day  observation  that  alcohol  and 
gas  are  formed  when  ordinary  yeast  grows  in  fruit  juice  or 
other  fluids  containing  sugar.  It  not  only  appears  that  bacteria 
sometimes  form  alcohol  and  gas  from  sugar,  but  that  with 
different  kinds  of  bacteria  and  different  kinds  of  food  material 
a  great  number  of  substances  are  formed,  some  of  which  are 
powerful  poisons.  In  most,  if  not  in  all,  of  the  diseases  caused 
by  bacteria  such  poisons  are  produced  within  the  living  body 
of  the  affected  individual,  and  the  symptoms  of  the  disease  and 
the  changes  produced  in  the  body  are  certainly  due  to  these 
poisons,  as  a  rule,  rather  than  to  the  direct  action  of  bacteria. 

On  account  of  their  extremely  minute  size,  the  bacteria  can- 

*  See  Part  II.,  Chapter  I. 


INTRODUCTION.  3 

not  be  seen  as  individuals  without  the  aid  of  the  microscope, 
although  great  numbers  of  them  taken  together  may  form  a 
plainly  visible  mass  of  growth.  When  they  are  examined 
with  the  microscope  they  appear  as  little,  round,  rod-shaped  or 
curved  bodies,  which  may  be  likened  to  so  many  periods,  dashes 
and  commas.  It  is  at  once  perceived  that  each  bacterium  is  an 
individual  by  itself,  and  that  it  consists  of  a  single  cell,  not  of  an 
aggregation  of  cells,  as  do  most  of  the  common  plants  and 
animals. 

Under  favorable  conditions  bacteria  undergo  rapid  multipli- 
cation. The  individuals  in  some  forms  divide  into  two  cells, 
in  other  forms  into  four  cells,  in  others  again  into  eight  cells 
simultaneously.  The  process  takes  place  by  direct  cell  divi- 
sion, and  is  called  fission". 

Under  certain  conditions,  bright,  glistening  bodies  make 
their  appearance  in  certain  bacteria,  and  become  larger  and 
larger,  while  the  cells  in  which  they  develop  break  up  into  fine 
fragments.  These  bodies  are  called  spores,  and  represent  a 
resting  stage  in  some  respects  resembling  the  seeds  of  higher 
plants.  They  have  much  greater  resisting  power  against 
injurious  influences  than  is  possessed  by  the  growing  or 
vegetative  forms.  There  are  spores  that  can  withstand  boiling 
for  hours,  but  fortunately  that  it  not  true  of  the  spores  of  any  of 
the  bacteria  that  produce  disease,  as  far  as  is  yet  known.  The 
earlier  investigators  observed  the  appearance  of  bacteria  in 
nutrient  infusions  which  they  had  endeavored  to  sterilize  by 
heat.  They  looked  upon  this  fact  as  indicating  the  possibility 
of  spontaneous  generation,  and  it  furnished  the  chief  support  of 
that  theory.  Probably  their  infusions  contained  very  resistant 
spores,  and  were  in  reality  not  sterile. 

Definition. — From  these  facts  a  definition  for  bacteria  may  be 
formulated. 

Bacteria  (Greek  paXTriptoi>,  meaning  a  little  stick)  are  ex- 
tremely minute,  unicellular  plants,  which  have  no  chlorophyll,  and 


4  MANUAL    OF    BACTERIOLOGY. 

which  divide  by  fission.  They  are  sometimes  called  schiz- 
omycetes.  In  every-day  language  they  are  known  as  microbes 
and  also  as  germs.  They  are  generally  classed  with  the  fungi. 
In  some  respects  they  seem  quite  closely  related  to  the  algae  or 
simplest  green  plants,  and,  on  the  other  hand,  they  have  strong 
points  of  likeness  with  some  of  the  unicellular  animals  belong- 
ing to  the  infusoria. 

Bacteria  are  divided  into  two  great  groups: 

I.  The  lower  bacteria  include  those  forms  which  are  of  most 
importance  at  present,  from  a  medical 
and  economic  point  of  view,  and  con- 


Sistof: 

Micrococci.  Bacilli.  Spirilla.         Cocco-bacilU—mostly    short,    thick, 
oval  forms  which  also  occur  in  rods. 

Micrococci,  or  cocci  (singular,  coccus)  —  spherical  forms. 

Bacilli  (sing.,  bacillus)  —  long  and  straight,  or  rod-shaped 
bacteria. 

Spirilla  (sing.,  spirillum)  —  consisting  of  spiral  filaments  like 
the  turns  of  a  corkscrew,  or  parts  of  spirals  shaped  like  commas. 

II.  The  higher  bacteria,  which  consist  of  long  filaments 
made  up  of  segments  more  or  less  united.  In  some  of  these  the 
filaments  show  dichotomous  branching.  This  group  is  more 
fully  discussed  under  the  non-pathogenic  bacteria,  Part  III. 
A  few  of  them  are  pathogenic. 

The  extreme  smallness  of  the  bacteria  is  hard  of  compre- 
hension. We  may  say,  of  most  of  them,  that  from  5,000  to 
25,000  placed  end  to  end  would  make  a  line  about  an  inch  in 
length.  The  tiny  speck  which  adheres  to  the  end  of  a  fine 
platinum  wire  when  this  is  used  to  obtain  preparations  from 
cultures  is  found.  upon  examination  with  the  microscope  to  con- 
sist of  many  thousands  of  bacteria. 

It  is  well  known  that  bacteria  are  present  on  most  of  the 
objects  about  us.  They  occur  on  the  skins  of  men  and  other 
animals  as  well  as  in  the  mouth,  stomach  and  intestines,  and 


INTRODUCTION.  5 

on  most  of  the  surfaces  of  the  body  that  open  to  the  external 
world.  They  are  found  in  the  water  of  rivers  and  lakes  and  in 
the  ocean.  They  appear  in  the  soil  down  to  a  depth  of  several 
feet.  They  float  in  the  air,  except  at  high  altitudes  and  over 
the  ocean.  Nansen  found  bacteria  on  the  ice  of  the  Polar  sea. 
Investigators  have  even  reported  finding  them  fossilized,  indi- 
cating, as  we  might  expect,  that  they  existed  at  remote  periods 
in  the  earth's  history.  But  the  vast  majority  of  them  are  en- 
tirely harmless  so  far  as  we  are  concerned,  and  many  of  them 
are  indispensable  in  maintaining  the  balance  existing  between 
dead  matter  and  living  beings. 

Were  it  not  for  the  putrefactive  and  nitrifying  bacteria,  the 
dead  bodies  of  plants  and  animals  would  lie  practically  un- 
changed where  they  fall,  and  the  fertilization  of  the  soil  neces- 
sary for  the  life  of  most  plants,  by  means  of  substances  derived 
from  such  dead  material,  would  cease. 

Some  kinds  of  bacteria  are  useful  in  taking  nitrogen  from  the 
atmosphere,  and  making  it  available  as  plant  food  in  the  soil, 
and  are  thus  employed  in  the  place  of  chemical  fertilizers. 
Many  of  them  have  been  made  to  subserve  a  useful  purpose  in 
the  ripening  of  cream  and  cheese,  and  in  the  manufacture  of 
vinegar  from  wine  and  cider.  It  has  been  suggested  with 
some  plausibility  that  anaerobic  bacteria  played  an  im- 
portant part  in  the  formation  of  coal  from  vegetable  substances. 

The  ripening  of  ensilage  in  silos  is  a  process  of  fermentation 
caused  by  bacteria. 

In  northern  Siberia  the  bodies  of  the  extinct  species  of  ele- 
phant called  mammoths  have  been  found  imbedded  in  frozen 
soil  where  they  appear  to  have  lain  for  thousands  of  years.  In 
this  case  the  growth  of  putrefactive  bacteria  has  been  prevented 
by  cold,  as  in  the  modern  refrigerator  or  cold-storage  plant. 

The  study  of  bacteria  has  led  to  the  understanding  of  many 
hitherto  unexplained  phenomena.  The  unaccountable  devel- 
opment of  a  moist,  brilliant  red  deposit  on  bread  and  other 


6  MANUAL    OF    BACTERIOLOGY. 

articles  of  food,  which  was  formerly  believed  by  the  super- 
stitious to  be  blood,  deposited  by  some  miraculous  agency,  we 
know  to  be  due  to  the  growth  of  a  common  organism  (bacillus 
prodigiosus).  The  emission  of  light  by  decaying  substances 
when  seen  in  the  dark  is  caused  by  bacteria  as  well  as  other 
organisms. 

It  seemed  that  in  some  cases  in  which  death  has  been  at- 
tributed to  the  suction  of  air  into  the  veins,  because  air  ap- 
peared to  be  present  inside  the  heart,  the  air  was  in  reality  a  gas 
formed  by  certain  bacilli  that  invaded  the  body  just  before  or 
just  after  death  (bacillus  aerogenes  capsulatus). 

Woodhead  tells  us  that  some  savages  are  in  the  habit  of 
smearing  the  soil  of  certain  localities  upon  their  arrows  for  an 
arrowpoison,  which  is  intelligible  in  the  light  of  the  fact  that 
soil  often  Contains  the  bacilli  of  tetanus  (lockjaw). 

The  comparatively  small  number  of  species  of  bacteria  that 
cause  disease  are  the  ones  that  interest  us  most,  and  are  those 
which  have  been  most  carefully  studied.  Since  the  bacteria  in 
common  with  other  fungi  are  compelled  to  derive  their  food 
from  organic  matter,  it  is  easy  to  understand  that  they  should 
frequently  exist  as  parasites  upon  living  animals  and  plants. 
Pear-blight  and  some  other  diseases  of  plants  are  caused  by 
bacteria.  Bees  and  other  insects,  frogs,  birds,  cattle  and  a 
great  number  of  animals  besides  men  suffer  from  diseases  pro- 
duced by  bacteria. 

When  bacteria  are  placed  upon  slips  of  glass  they  may  be 
studied  with  the  microscope  while  alive.  Some  of  them  when 
living  are  motionless;  others  wriggle  vigorously.  Some  dart 
about  like  minnows  in  a  stream,  or  they  make  their  way  slowly 
across  the  field  of  the  microscope  like  a  boat  that  is  being 
sculled  from  the  stern.  By  proper  methods  it  can  be  shown 
that  the  movements  are  effected  through  one  or  more  fine, 
hair-like  processes  called  flagella. 

Often  it  is  expedient  to  study  bacteria  after  drying  them  on 


INTRODUCTION.  7 

slips  of  glass,  when  they  may  be  made  more  conspicuous  by 
giving  them  an  artificial  color  (staining) .  Some  of  the  substances 
of  which  they  are  composed  readily  absorb  certain  dyes.  For 
this  purpose  the  aniline  dyes  are  used,  and  their  employment 
has  been  one  of  the  important  factors  in  making  progress  in 
bacteriology  possible. 

With  the  microscope  alone  it  is  not  usually 
practicable  to  distinguish  accurately  between 
various,  kinds  of  bacteria.  Micrococci,  for  in- 
stance, which  are,  in  reality,  entirely  different 
species,  may  look  very  much  alike.  But 
differences  usually  become  apparent  when  the 
bacteria  are  grown  artificially.  The  cultiva- 
tion is  done  for  the  most  part  in  test-tubes 
containing  some  material  which  furnishes 
suitable  food.  The  nutrient  materials  most 
used  are  meat-extract  and  peptone,  which, 
dissolved  with  salt  in  water,  constitute  nutrient 
bouillon.  Ordinary  gelatin,  or  a  vegetable 
gelatin  called  agar-agar,  may  be  added  to  the 
bouillon  when  a  solid  culture-medium  is  de- 
sired. Before  these  substances  can  be  used 
for  the  cultivation  of  bacteria  all  other  bac- 
teria which  they  may  contain  must  be  de- 

'  J  FIG.    2. — Test- 

Stroyed  by  heat.  tube      containing 

Finally,  the  effects  of  bacteria  in  bringing     cul^re-medium. 
about  disease  may  be   tested   on   the   lower 
animals.      The  proof  that   a  particular  species   of   bacteria 
causes  a  particular  disease  cannot   be   considered   complete 
unless  the  disease  can  be  reproduced  by  introducing  these 
bacteria  into  some  animal. 

Bacteriological  Literature. — The  student  who  wishes  to 
pursue  bacteriological  study  in  any  direction  farther  than  it  is 
possible  for  the  limits  of  a  short  manual  to  go,  may,  besides 


8  MANUAL    OF    BACTERIOLOGY. 

consulting  the  large  text-books  and  weekly  medical  journals, 
obtain  much  assistance  from  technical  periodicals.  The 
Journal  oj  Experimental  Medicine,  Journal  of  Medical  Research, 
and  the  Journal  of  Infectious  Diseases,  published  in  this  country, 
and  the  English  Journal  of  Pathology  and  Bacteriology  and 
Journal  oj  Hygiene  devote  much  valuable  space  to  the 
subject. 

The  Journal  of  Tropical  Medicine  also  contains  contribu- 
tions. 

A  reading  knowledge  of  German  and  French  is  very  desirable. 
The  Centralblatt  fur  Bakteriologie,  etc.,  a  German  periodical, 
and  the  Bulletin  de  VInstitut  Pasteur,  published  semimonthly 
in  Paris,  contain  abstracts  of  most  of  the  important  researches 
made  in  all  parts  of  the  world.  The  Annales  de  Vlnstitut 
Pasteur,  the  Zeitschrijt  fur  Hygiene,  the  Archiv  fur  Hygiene 
and  the  Hygienische  Rundschau  contain  many  original  arti- 
cles on  bacteriological  subjects. 

The  whole  literature  of  any  specified  subject  in  bacteriology 
can  be  most  conveniently  found  in  Baumgarten 's  Jahresbericht 
der  Mikroorganismenlehre. 

Those  who  are  interested  in  agricultural  bacteriology  should  read  the  ex- 
periment station  records  and  the  various  bulletins  issued  by  the  Department 
of  Agriculture  of  the  United  States.  They  can  usually  be  obtained  upon  appli- 
cation to  the  Department  at  Washington,  D.  C.  The  bacteria  that  produce 
disease  in  domestic  animals  are  described  in  Dr.  V.  A.  Moore's  book,  "The 
Infectious  Diseases  of  Animals,"  Taylor  &  Carpenter,  Ithaca,  N.  Y.,  1906,  and 
in  the  "Special  Report  on  the  Diseases  of  Cattle,"  United  States  Department  of 
Agriculture,  1904;  also  Diseases  of  the  Horse,  Department  of  Agriculture,  1907 

Historical  Sketch. — Nearly  all  that  we  know  of  bacteria 
and  the  part  they  play  in  producing  disease  has  been  learned 
during  the  last  half  of  the  last  century.  Nevertheless,  many 
facts  were  known  long  ago,  and  even  by  the  ancients,  which 
were  effective  in  directing  the  thought  of  later  years.  The 
epidemic  nature  of  certain  maladies  was  naturally  among 
the  earliest  of  these  to  be  noticed,  and  was,  even  until  com- 


INTRODUCTION.  9 

paratively  recent  times,  attributed  to  the  influence  of  gods, 
demons  or  other  supernatural  agencies.  The  superstitions 
and  crude  beliefs  of  the  past  gave  rise  to  a  mass  of  gro- 
tesque theories  and  fanciful  speculations.  But  with  all  this 
we  hear  of  certain  beliefs  and  practices  which  plainly  fore- 
shadowed those  of  the  present  day.  Latin  writers  nearly 
two  thousand  years  ago  recorded  a  relation  between  insects 
and  malaria  which  has  but  lately  been  proved  and  explained. 
The  isolation  of  lepers  by  the  ancient  Hebrews  shows  that  the 
infectious  character  of  the  disease  has  long  been  recognized, 
though  other  affections  than  leprosy  were  probably  confused 
with  this  disease  by  the  ancients.  "He  is  unclean:  he  shall 
dwell  alone;  without  the  camp  shall  his  habitation  be" 
(Lev.  XIII. ,  46).  There  is,  in  fact,  much  in  the  laws  of 
Moses  that  points  to  some  knowledge  of  the  nature  of  infec- 
tions. "This  is  the  law,  when  a  man  dieth  in  a  tent:  all  that 
come  into  the  tent  and  all  that  is  in  the  tent  shall  be  unclean 
for  seven  days.  And  every  open  vessel  which  has  no  covering 
upon  it  shall  be  unclean"  (Numb.  XIX.,  14,  15). 

"Everything  that  may  abide  the  fire,  ye  shall  make  it  go 
through  the  fire,  and  it  shall  be  clean"  (Numb.  XXXL,  23). 

In  Homer  we  read  of  Ulysses,  that,  having  slain  his  wife's 
troublesome  suitors: 

"With  fire  and  sulphur,  cure  of  noxious  fumes, 
He  purged  the  walls  and  blood-polluted  rooms"  (Pope's  Odyssey). 

The  massive  aqueducts  of  the  Romans  still  remain  to  testify 
that  they  understood  the  importance  of  a  pure  water-supply. 

In  Rome  there  were  also  sewers  for  the  disposal  of  drainage, 
while  the  Cretans  and  Assyrians  used  sewerage  systems  hun- 
dreds and  even  thousands  of  years  before. 

About  the  fourteenth  century  we  find  quarantine  against 
infectious  diseases,  plague  in  particular,  practiced  by  certain 
Italian  cities;  and  the  word  "quarantine"  came  into  use  from 


10  MANUAL    OF    BACTERIOLOGY. 

the  fact  that  the  period  of  detention  was  about  forty  days 
(Ital.  quarantina).* 

Leeuwenhoek,  a  citizen  of  Delft,  in  Holland  (1632-1723) 
appears  to  have  been  the  first  who  actually  saw  bacteria. 
Yeast-cells  he  certainly  observed,  besides  making  many  other 
contributions  of  great  value  to  biology.  Leeuwenhoek  pro- 
duced admirable  lenses  of  high  magnifying  power,  and  de- 
scribed what  he  witnessed  with  singular  accuracy  and  en- 
thusiasm. 

Even  before  this  time  men  had  sought  to  explain  the  phenom- 
ena of  infectious  diseases  by  supposing  the  body  to  have  been 
penetrated  by  minute  parasites — for  example,  worms.  The 
spread  of  such  diseases  through  a  community  from  a  single 
center  could  readily  be  accounted  for  by  the  multiplication  of 
a  contagious  element,  itself  alive  (contagium  vivum).  With  in- 
creasing knowledge  of  the  abundance  of  microscopic  life  these 
speculations  took  firmer  hold.  But  long  before  their  truth  was 
finally  demonstrated  great  advances  were  made  in  the  preven- 
tion of  infectious  diseases.  Much  honor  is  due  the  clinicians, 
whose  accurate  observations  and  foresight  accomplished  im- 
portant results  at  an  early  day,  working  with  what  now  seems  a 
very  meagre  knowledge  of  the  facts. 

The  production  of  immunity  from  small-pox  by  inoculation 
was  first  practiced  in  oriental  countries.  The  method  had  long 
been  in  use  in  the  East,  when  in  1718  it  was  brought  to  the 
notice  of  Europeans  by  Lady  Montagu,  wife  of  the  English 
ambassador  at  Constantinople.  The  procedure  consisted 
simply  of  the  introduction  of  the  virus  of  small-pox  by  puncture 
of  the  skin.  An  attack  of  small-pox  resulted,  which  was 
usually  much  milder  and  far  less  dangerous  than  the  natural 
disease. 

Lady  Montagu  stated  in  a  letter:  "Every  year  thousands 

*J.  M.'  Eager.  The  Early  History  of  Quarantine.  Yellow  Fever  Institute 
Bulletin,  No.  12.  U.  S.  Marine  Hospital  Service. 


INTRODUCTION. 


II 


undergo  the  operation;  and  the  French  ambassador  says  pleas- 
antly that  they  take  the  small-pox  here  by  way  of  diversion,  as 
they  take  the  waters  in  other  countries."  The  mild  attacks 
that  followed  inoculation  were,  however,  just  as  contagious  to 
other  persons  as  the  natural  disease,  so  that  the  dangers  of  this 
practice  to  the  community  were  very  great. 

This  was  previous  to  the  introducion  of  vaccination  by 
Edward  Jenner  in  1796.  At  this  time  a  belief  was  current 
among  farmers  that  a  mild  form  of  disease,  called  cow-pox, 
acquired  by  milkers,  furnished  protection  against  small-pox, 
and  on  investigation  Jenner  found  this  belief  to  be  justified. 
In  a  few  years  the  practice  of  vaccination  spread  to  all  parts 
of  the  world.* 

It  was  introduced  into  the  United  States  by  Dr.  Benjamin 
Waterhouse,  of  Harvard.  President  Thomas  Jefferson  was 
active  in  bringing  it  into  general  use,  especially  in  the  South. 

The  infectious  nature  of  puerperal  fever  was  first  demon- 
strated by  Semmelweis,  of  Vienna,  in  1847.  Before  this  time 
unsuccessful  attempts  had  been  made  to  prove  that  atmospheric 
influences  were  responsible  for  the  disease,  and  during  the 
seventeenth  and  eighteenth  centuries  the  course  had  been 
supposed  to  lie  in  the  absorption  of  the  milk  from  the  breasts 
into  the  blood.  But  Semmelweis  was  struck  by  the, similarity 
between  puerperal  fever  and  a  fatal  case  of  pyemia  following  a 
dissecting  wound  in  the  case  of  a  friend  of  his,  and  was  led  by 
this  observation  to  attribute  the  origin  of  the  disease  to  poisons 
carried  on  the  fingers  of  physicians  and  students  from  the 
dissecting-room  to  the  woman  in  childbed.  This  idea  of 
Semmelweis  aroused  opposition  and  ridicule,  but  it  withstood 
these  tests  though  the  originator  somewhat  modified  his 
views.  As  a  prophylactic  measure,  Semmelweis  advocated 

*See  the  works  of  Edward  Jenner  by  Dock.  New  York  Medical  Journal 
Nov.  29  and  Dec.  6,  1902.  Also  Dulles.  The  History  of  Vaccination.  Phila 
delphia  Medical  Journal.  May  30,  1903. 


12  MANUAL    OF    BACTERIOLOGY. 

washing  the  hands  of  the  attendant  at  child-birth  in  solutions 
of  chlorine  or  chloride  of  lime  in  addition  to  cleansing  them 
with  soap  and  water. 

During  the  same  period  similar  ideas  were  advanced  by  Dr. 
Oliver  Wendell  Holmes  in  the  United  States.  His  paper  on 
"The  Contagiousness  of  Puerperal  Fever"  appeaed  in  1843. 
A  lively  controversy  lasting  several  years  was  provoked,  in 
which  Holmes  defended  his  position  with  great  vigor.  His 
admirable  literary  style  served  him  effectively.* 

In  the  first  half  of  the  nineteenth  century,  with  improved 
microscopes,  knowledge  of  minute  living  things  grew  rapidly, 
chiefly  with  respect  to  infusoria  and  other  relatively  large 
forms.  In  1840  Henle  described  the  part  played  by  micro- 
organisms in  producing  disease  in  terms  surprisingly  in  accord 
with  views  held  at  the  present  time.  His  deductions  were 
based  almost  entirely  on  knowledge  of  the  general  -nature, 
spread  and  course  of  infections.  So,  too,  Villemin  anticipated 
the  discovery  of  the  bacillus  of  tuberculosis,  for  he  transmitted 
the  disease  to  animals,  by  inoculating  them  with  material  from 
cases  of  tuberculosis  in  man. 

The  key  to  exact  knowledge  of  the  microorganisms  of  disease 
was  finally  discovered  in  the  study  of  fermentation.  No 
better  illustration  could  be  found  of  the  possible  value  to 
mankind  which  may  lie  in  any  addition  whatever  to  the  com- 
mon stock  of  knowledge.  The  study  of  bottles  of  bad-smelling 
broth  would  have  seemed,  fifty  years  ago,  a  most  unpromising 
beginning  for  the  discovery  of  the  causes  of  cholera,  plague 
and  the  like,  or  for  an  antitoxin  for  diphtheria. 

Studies  on  Fermentation  and  Spontaneous  Generation. — Two 
observers  (Schwann,  Cagniard-Latour,  1837)  almost  simul- 
taneously advanced  the  idea  that  yeast-cells  were  living  organ- 
isms, and  that  the  fermentation  of  solutions  of  sugar  was  due 
to  their  growth.  This  amused  a  controversy  which  lasted  more 

*  See  O.  W.  Holmes.     Medical  Essays.     Houghton,  Mifflin  &  Co.,  1889. 


INTRODUCTION.  13 

than  thirty  years.  Prominent  among  those  who  contended 
against  the  agency  of  living  cells  in  the  production  of  fermenta- 
tion was  Liebig,  but  in  spite  of  this  the  doctrine  steadily  gained 
ground,  and  was  extended  to  include  other  sorts  of  fermenta- 
tion and  the  putrefaction  of  albuminous  material.  Different 
•kinds  of  fermentation,  with  different  products,  such  as  acetic 
acid  and  butyric  acid,  were  eventually  shown  to  be  due  to  the 
growth  of  different  kinds  of  microbes. 

These  microbes  were  found  to  be  fungi  of  various  sorts,  and 
chiefly  one  or  another  variety  of  bacteria.  The  most  cele- 
brated among  the  students  of  fermentation  was  Pasteur,  the 
simplicity  and  kindness  of  whose  character  excite  our  admira- 
tion equally  with  his  devotion  to  his  work.* 

Before  the  nature  of  fermentation  was  understood,  the  pos- 
sibility of  spontaneous  generation  had  been  universally  ad- 
mitted. When  vermin  of  various  sorts  appeared  in  putrefying 
material  the  conclusion  was  drawn  that  they  had  their  origin 
directly  from  it.  Although  that  had  long  since  been  disproved 
in  the  case  of  large  organisms  like  worms  and  frogs,  still,  as 
late  as  the  middle  of  the  last  century,  it  was  held  by  many  to 
account  for  the  swarming  microscopic  life  found  in  fermenting 
fluids.  A  flask  of  meat  broth  left  exposed  to  the  air  will  after 
a  few  days  contain  countless  tiny  living  things,  chiefly  bacteria. 
Pasteur  and  his  followers  showed  that  these  bacteria  were  the 
progeny  of  others  already  in  the  flask  or  which  had  fallen  in 
from  the  air. 

When  the  flask  of  broth  was  boiled,  no  development  of  or- 
ganisms took  place,  if  the  entrance  of  germs  from  the  atmos- 
phere was  prevented.  The  latter  was  accomplished  by  such 
devices  as  heating  the  air,  passing  it  through  sulphuric  acid, 
using  a  flask  with  a  long  twisted  neck  or  by  plugging  the  flask 
with  cotton  (Schroder  and  Von,  Dusch). 

*See  Louis  Pasteur.  His  Life  and  Labors.  By  His  Son-in-Law.  Translated, 
by  Lady  Claude  Hamilton. 


14  MANUAL    OF    BACTERIOLOGY. 

To  prove  that  boiling  had  not  made  the  fluid  unfit  for  the 
growth  of  organisms,  air  was  subsequently  allowed  to  have 
access  to  it  without  such  precautions,  when  putrefaction  took 
place  in  the  usual  manner. 

These  principles  underlie  the  methods  used  daily  for  the 
preservation  of  meat,  fruit  and  vegetables,  in  the  household  and 
in  canning  factories. 

Although  boiling  occasionally  failed  to  prevent  fermentation, 
investigators  came  with  practice  to  have  a  smaller  number  of 
failures.  Such  failures  it  was  shown  were  due  to  the  presence 
of  the  resistant  forms  of  the  organisms  called  spores  previously 
alluded  to  which  some  bacteria  assume.  The  true  nature  of 
spores  was  recognized  later  by  Cohn.  Pasteur  found  that 
exposure  to  steam  at  temperatures  sufficiently  high  above  the 
boiling  point  would  destroy  the  most  resistant  microbes  and 
their  spores.  But  even  boiling  and  subsequent  protection 
from  the  entrance  of  bacteria  sometimes  met  with  failure. 

The  controversies  over  fermentation  and  putrefaction  lasted 
almost  until  the  present  day.  They  have  been  productive  of 
numerous  benefits  to  the  arts  and  manufactures.  But,  what  is 
of  more  importance  to  our  subject,  they  led  to  a  vastly  better 
understanding  of  diseases  produced  by  microorganisms.  The 
study  of  bacteria  has  been  pursued  with  such  vigor  in  the 
last  thirty-five  years  in  fact  that  most  of  what  we  know  con- 
cerning the  bacteria  of  disease  has  been  learned  during  this 
period,  and  advances  are  still  constantly.being  made. 

The  discussions  concerning  fermentation  and  putrefaction 
were  still  going  on  when  Lister  made  his  brilliant  deduction 
that  suppuration  and  septic  processes  in  wounds  were  a  species 
of  fermentation  (1867).  From  this  came  the  antiseptic  and 
aseptic  methods  of  operating  and  of  dressing  wounds,  which 
have  made  possible  the  wonderful  results  of  modern  operative 
surgery.* 

*See  Roswell  Park.     History  of  Medicine. 


INTRODUCTION.  15 

In  1834  the  parasite  of  itch  (Acarus  scabiei,  the  itch  mite,  an 
arachnid,  related  to  the  insects)  was  discovered,  and  the  cause 
of  one  contagious  malady  determined. 

Quite  early  in  the  nineteenth  century  also  the  relatively  large 
fungi  of  thrush  and  some  of  the  parasitic  skin  diseases  were 
discovered.  The  bacilli  of  anthrax,  which  are  also  relatively 
large,  were  seen  in  the  blood  of  animals  by  Pollender  in  1855 
and  Davaine  in  1863. 

Davaine  produced  anthrax  in  animals  by  injecting  into  them 
blood  containing  anthrax  bacilli.  But  complete  proof  that 
these  bacilli  were  the  cause  of  the  disease  required  that  they 
should  produce  it  when  injected  alone  and  when  freed  from  the 
smallest  trace  of  material  derived  from  the  first  diseased 
animal.  Unless  these  conditions  were  complied  with,  some 
other  material,  for  example  an  enzyme  or  ferment,  might  be 
supposed  to  be  carried  from  the  first  to  the  second  animal  and 
to  be  the  real  cause  of  the  disease.  For  this  purpose  it  was 
necessary  to  cultivate  the  bacilli  outside  the  animal  body  in  an 
artificial  medium  of  some  kind,  such  as  meat  broth,  as  was 
done  by  Pasteur.  It  then  became  possible  to  demonstrate 
that  their  properties  could  remain  unaltered  after  being  grown 
in  successive  generations  on  different  lots  of  broth.  As  bacteria 
of  two  or  three  species  were  often  encountered  in  mixtures,  it 
became  most  important  to  secure  a  method  by  which  the  dif- 
ferent species  could  be  separated  from  one  another  and  be 
propagated  as  separate  "pure  cultures."  This  was  done  suc- 
cessfully by  diluting  such  mixtures  greatly,  so  that  a  drop 
planted  in  a  new  tube  of  broth  should  contain  only  a  single 
organism.  The  growth  ensuing  would  of  course  consist  of  the 
same  kind  of  organism  exclusively.  Such  procedures  were 
uncertain  and  very  laborious. 

Koch  introduced  in  1881  his  method  of  separating  bacteria 
by  "plating,"  described  below  (Part  L,  Chapter  V.),  and  this 
is  probably  the  most  important  contribution  to  bacteriological 


1 6  MANUAL    OF    BACTERIOLOGY. 

technic  which  has  ever  been  made.  Koch  also  pointed 
out  the  advantages  of  solid  media  for  the  propagation  of 
pure  cultures.  Other  important  technical  improvements  of 
the  same  period  were  the  adoption  of  the  illuminating  apparatus 
of  Abbe  and  immersion  objectives,  and  of  aniline  dyes  for 
staining  bacteria  and  making  them  visible  (Weigert  and 
Ehrlich).  Beginning  with  the  bacillus  tuberculosis  described 
by  Koch  in  1882,  a  number  of  pathogenic  bacteria  were  dis- 
covered during  the  ensuing  years  in  rapid  succession. 

The  application  of  the  newly-gained  knowledge  concerning 
the  bacteria  causing  infectious  diseases  to  the  prevention  and 
cure  of  these  diseases  was  begun  almost  immediately  by  Pasteur. 
A  few  facts  existed  to  guide  the  direction  of  the  research.  It 
had  been  known  even  in  ancient  times  that  one  attack  of  an 
infectious  disease,  such  as  scarlet  fever,  may  confer  immunity 
from  subsequent  attacks. 

The  protection  against  small-pox  which  was  furnished  by 
vaccination  also  was  suggestive,  although  the  mechanism  by 
which  this  protection  came  about  was  not  understood. 

Pasteur  worked  on  the  theory  that  immunity  from  a  disease 
would  probably  be  secured  by  producing  a  mild  attack  of  the 
disease.  Such  a  mild  attack  might  be  expected  to  follow  if  a 
susceptible  individual  were  inoculated  with  microbes  of  lowered 
virulence.  Various  methods  were  employed  to  reduce  the 
virulence  of  bacteria,  notably  cultivation  at  high  temperatures 
(43°C.).  Pasteur  was  able  to  produce  immunity  against  a 
number  of  the  diseases  of  the  lower  animals.  His  method  of 
inoculating  sheep  and  cattle  against  anthrax  has  been  employed 
with  some  success.  A  somewhat  similar  principle  has  led  to 
the  preparation  of  a  vaccine  for  the  disease  of  cattle  called 
"black  leg,"  and  such  vaccine  is  now  distributed  gratuitously 
to  farmers  by  the  United  States  government.  Inoculation  of 
human  subjects  with  the  attenuated  virus  is  used  for  hydro- 
phobia. This  method  also  was  devised  by  Pasteur. 


INTRODUCTION.  1 7 

But  not  only  have  inoculations  against  the  microorganisms 
themselves  been  perfected  in  some  cases,  but  also  the  sub- 
stances which  are  called  antitoxins,  the  specific  agents  against 
the  poisonous  products  of  the  bacteria,  have  been  produced. 
The  discovery  of  these  antitoxins  for  infectious  diseases 
(see  Part  II. ,  Chapter  VII.)  we  owe  to  Behring.  This  portion 
of  our  subject  belongs  entirely  to  the  present  day,  and  is  now 
being  studied  with  great  energy. 

Allusion  has  already  been  made  to  moulds  and  other  micro- 
scopic parasites  whose  nature  makes  their  study  almost  in- 
separable from  that  of  the  bacteria.  In  this  class  also  belong 
the  'primitive  forms  of  animal  life,  the  Protozoa,  which  are  the 
causes  of  amebic  dysentery  (Losch,  1875)  and  malaria  (Lav- 
eran,  1880).  The  disease  of  cattle  called  "Texas  fever"  is  also 
caused  .by  a  protozoon.  Theobald  Smith  in  the  United  States 
discovered  that  the  parasite  of  Texas  fever  is  conveyed  from 
one  animal  to  another  by  the  cattle-tick.  Since  then  it  has 
been  shown  (by  Manson,  Ross  and  others)  that  malaria  is 
conveyed  by  mosquitoes  from  a  person  having  the  disease  to 
one  not  affected.  It  has  also  been  shown  by  Reed  and 
Carroll  that  a  similar  relation  exists  between  mosquitoes  and 
yellow  fever.  The  part  played  by  flies  and  other  insects  in 
carrying  disease  germs  is  still  receiving  active  attention  and  the 
future  may  show  that  these  play  a  most  important  part  in 
diseases  other  than  those  already  mentioned'. 

In  1903  Novy  and  McNeil  succeeded  in  making  pure  cul- 
tures of  pathogenic  protozoa  grow  in  tubes,  in  nearly  the  same 
way  that  cultures  of  bacteria  are  propagated  (see  Appendix). 

It  is  encouraging  to  reflect  that  the  progress  of  bacteriology 
has  been  made  by  gradual  and  logical  steps.  The  great  dis- 
coveries have  not  been  lucky  accidents,  but  have  been  worked 
out  patiently  and  with  deliberation. 


PART  I. 


CHAPTER  I. 

EXAMINATION    OF    BACTERIA   WITH    THE    MICROSCOPE, 
INCLUDING    METHODS  OF  STAINING. 

The  Microscope. — The  microscope  consists  of  a  tubular 
body  which  carries  the  optical  parts,  and  which  can  be  raised  or 
lowered  for  focusing.  It  is  a  matter  of  convenience  to  have 
three  lenses  attached  to  the  body  of  the  instrument  by  means 
of  a  triple  nose-piece,  which  permits  any  objective  to  be  turned 
into  the  optical  axis  at  will.  But  a  low-power  dry  lens  and  an 
oil-immersion  objective  are  all  that  are  essential  for  studying 
the  bacteria.  The  eye-piece  slips  into  the  upper  and  opposite 
end  of  the  body  or  tube.  The  arrangements  for  focusing  con- 
sist of  a  rack  and  pinion  which  accomplish  the  coarse  adjust- 
ment, 'and  a  more  delicate  fine  adjustment.  The  stage,  upon 
which  the  objects  to  be  examined  are  placed,  has  an  opening  in 
the  middle.  In  this  opening  an  iris  diaphragm  and  Abbe 
condenser  are  inserted.  The  iris  diaphragm  enables  one  to 
alter  the  size  of  the  opening  as  desired.  Beneath  the  stage  is  a 
movable  mirror,  of  which  one  side  is  plane  and  the  other 
concave.  All  of  these  parts  are  supported  on  a  short,  heavy 
pillar,  which  is  fixed  in  the  horseshoes-shaped  base. 

The  essential  parts  of  the  microscope  are,  of  course,  the  eye- 
piece (German,  Ocular)  and  the  objective.  Objectives  are 
variously  designated  by  different  makers,  for  instance,  some 
use  letters,  A,  B,  C,  etc.,  others  use  numbers,  i,  2,  3,  etc. 
others  again  give  the  focal  distance,  as  f  inch,  J  inch,  J  inch, 

18 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         19 

etc.  In  bacteriological  work  a  rather  "low-power"  §  or  j-inch 
objective,  and  a  high-power  TV  inch  oil-immersion  objective 
are  needed.  A  J  or  J  inch  dry  objective  may  also  occasionally 


FIG.  3. — Microscope. 

be  useful.  The  magnification  with  the  f  or  f-inch  objective  is 
about  75  to  100  diameters;  with  the  J  to  J-inch  300  to  5oc 
diameters;  with  the  y1-^  immersion  750  to  1000  diameters.  The 
magnification  varies  according  to  the  eye-piece  used,  as  well 


20 


MANUAL  OF  BACTERIOLOGY. 


as  with  the  objective.  A  i-inch  and  ij-inch  eye-piece  (Zeiss 
No.  2  and  No.  4)  serve  well  for  most  purposes.  The  eye- 
pieces are  usually  named  arbitrarily,  like  the  objectives.  In 
using  the  TV  immersion  objective  a  layer,  of  thickened  oil  of 
cedarwood  is  placed  between  the  lower  surface  of  the  objective 
and  the  upper  surface  of  the  glass  covering  the  object  under 
examination.  The  oil  must  be  wiped  away  from  the  surface 
of  the  objective  when  the  examination  is  finished.  For  this 
purpose  the  soft  paper  sold  by  dealers  in  microscopic  ap- 
paratus serves  admirably.  Care  must  be  taken  not  to  scratch 
the  lower  surface  of  this  objective.  Oil  of  cedar- wood  furnishes 


FIG.  4. — Abbe  condenser. 
On  the  right  side  the  figure  gives  a  sectional  view. 


a  medium  having  nearly  the  same  refractive  index  as  the  glass 
of  the  lens  as  the  glass  on  which  the  object  is  mounted,  and  it 
obviates  the  dispersion  of  light  which  takes  place  when  a  layer 
of  air  is  interposed  between  the  objective  and  the  object,  as 
happens  with  the  ordinary  dry  lens.  This  object  is  used  in 
connection  with  the  Abbe  condenser,  which  consists  of  two 
or  three  lens  combined  so  as  to  focus  the  rays  coming  from  the 
plane  mirror  upon  the"  object.  The  condenser  gives  a  very 
intense  illumination  over  a  very  small  field.  The  condenser  is 
not  necessary  excepting  with  the  oil-immersion  objective.  If 
it  is  used  with  the  other  objectives,  the  illumination  must  be 
regulated  by  lowering  the  condenser,  closing  the  diaphragm 
more  or  less,  and  substituting  the  concave  for  the  plane  mirror. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         21 

It  is  to  be  remembered  that  more  depends  upon  securing  a 
distinct  picture  than  upon  a  very  high  magnification  of  the 
object. 

The  microscope  should  be  placed  in  front  of  the  observer 
on  a  firm  table  with  the  light  in  front.  The  observer  should 
be  able  to  bring  the  eye  easily  over  the  eye-piece  when  the  tube 
of  the  microscope  is  in  vertical  position.  Daylight  should  be 
employed  if  possible,  but  not  direct  sunlight.  When  artificial 
illumination  is  necessary,  an  ordinary  lamp,  a  Welsbach  burner 
or  an  incandescent  electric  light  may  be  used.  It  is  best  to 
modify  the  artificial  light  by  inserting  a  sheet  of  blue  glass 
between  the  light  and  the  mirror. 

In  order  to  focus  upon  any  object,  having  first  secured  a 
satisfactory  illumination  with  the  mirror,  it  is  best,  beginning 
with  the  low  power  and  using  the  coarse  adjustment  for  focus- 
ing, to  'bring  the  objective  quite  close  to  the  object,  and  then, 
with  the  eye  in  position,  to  raise  the  tube  until  the  object 
comes  into  focus.  The  exact  focusing  is  done  with  the  fine 
adjustment.  The  observer  should  keep  both  eyes  open  when 
using  the  microscope,  and  should  be  able  to  use  either  eye  at 
will. 

All  measurements  of  microscopic  objects  are  expressed  in 
terms  of  a  micromillimeter.  This  is  one-thousandth  of  a 
millimeter  (o.ooi  mm.),  which  is  about  -gr-Jinj- of  an  inch.  It 
is  generally  called  a  micron  for  short,  and  is  denoted  by  the 
Greek  letter  /a.  For  example,  5  ^  =  0.005  mm.  =  -j^nnr  mcn- 

The  Preparation  of  Specimens  of  Bacteria  for  Exami- 
nation with  the  Microscope. — The  substance  under  ex- 
amination is  placed  upon  thin  slips  of  glass  called  cover- 
glasses.  The  material  is  spread  over  the  cover-glass  by  means 
of  a  platinum  wire  which  has  been  fixed  in  a  glass  rod  about 
six  inches  long.  Such  a  platinum  wire  is  used  constantly  in 
doing  bacteriological  work.  It  is  the  tool  by  means  of  which 
one  is  able  to  handle  bacteria  with  impunity.  The  platinum 


22  MANUAL  OF  BACTERIOLOGY. 

wire  must  be  stiff  enough  not  to  bend  too  easily,  and  yet  it 
should  not  be  so  large  that  it  will  not  cool  rapidly  after  heating. 
A  good  size  for  most  purposes  is  No.  23,  American  wire 
gauge  (Brown  &  Sharp).  The  wire  may  be  straight  through- 
out its  length,  or  the  tip  may  be  bent  to  form  a  loop  (German, 
Oese).  It  is  well  to  follow,  from  the  beginning,  certain  rules 
which  make  the  use  of  the  platinum  wire  safe  and  accurate. 
Every  time  it  is  taken  into  the  hand  and  before  using  it  for  any 
manipulation  heat  it  in  the  flame  of  a  Bunsen  burner  or  an 
alcohol  lamp  to  a  red  heat;  and  always,  after  using,  and  before 
putting  it  down,  heal  it  again  to  a  red  heat.  If  the  needle  is  wet 
it  should  be  dried  by  holding  it  near  the  flame  in  order  to  avoid 


FIG.  5. — Straight  platinum  wire  and  platinum  loop."" 

the  "sputtering"  which  occurs  if  it  is  plunged  at  once  into  the 
flame.  This  precaution  is  especially  called  for  when  the  wire 
has  been  dipped  in  milk  or  other  substances  containing  oil. 
When  the  needle  "sputters,"  as  it  is  called,  from  too  rapid 
heating,  particles  that  have  not  yet  been  sterilized  may  be 
thrown  some  distance.  On  no  account  should  the  needle 
touch  any  object  other  than  that  which  it  is  intended  it  should 
touch.  With  such  a  platinum  wire,  which  has  been  properly 
sterilized,  one  can  easily  remove  portions  from  a  culture  of 
bacteria,  or  from  a  fluid  in  which  bacteria  are  supposed  to  be 
present.  The  glass  rod  in  which  the  platinum  wire  is  fixed 
should  be  held  between  the  thumb  and  forefinger  of  the  right 
hand  like  a  pen.  (For  the  manner  of  holding  test-tubes,  see 
page  79.) 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        23 

The  Hanging-drop. — Living  bacteria  may- be  studied  with 
the  microscope  while  suspended  in  some  fluid  substance. 
This  is  accomplished  by  means  of  a  hanging-drop.  In 
order  to  prepare  a  hanging-drop  for  examination  a  clean 
cover-glass  is  held  in  the  forceps  and  a  small  drop  of  the  fluid 
to  be  examined  is  spread  thinly  over  the  center  of  it  by  means 
of  a  platinum  needle  which  has  just  previously  been  heated  in  a 
flame  and  allowed  to  cool.  The  needle  should  again  be 
sterilized  in  the  flame.  When  cultures  on  solid  media  are  to 
be  examined,  a  small  particle  may  be  mixed  with  a  drop  of 
'sterilized  physiological  salt  solution  or  bouillon  which  has  first 
been  placed  in  the  middle  of  the  cover-glass.  The  cover-glass 
should  have  been  carefully  cleaned  and  sterilized  over  the 


FIG.  6. — Diagram  of  the  hanging-drop. 

flame.  The  cover-glass  with  the  thin  drop  of  fluid  material 
held  in  sterilized  forceps  is  now  to  be  inverted  over  a  sterilized 
glass  slide,  which  has  a  concavity  ground  in  the  middle  of  it. 
Around  the  concavity,  the  slide  should  be  smeared  with 
vaseline.  In  this  manner  a  small,  air-tight  chamber  is  made. 
This  preparation  may  be  put  upon  the  stage  of  the  micro- 
scope. A  good  dry  lens,  if  of  sufficiently  high  power,  is  more 
convenient  for  examining  the  hanging-drop  than  an  oil-im- 
mersion. If  the  latter  be  used,  having  placed  a  drop  of  cedar- 
oil  on  the  center  of  the  cover-glass,  and  a  good  light  having 
been  secured,  the  oil-immersion  objective  should  be  brought 
down  upon  this  drop  of  oil.  The  beginner  often  experiences 
difficulty  in  focusing  upon  a  hanging-drop.  It  is  well  to  shut 
off  most  of  the  light  by  means  of  the  iris  diaphragm.  Often  it 
is  well  to  secure  the  focus  roughly  upon  the  extreme  outer  edge 
of  the  chamber,  or  to  find  the  edge  of  the  drop  of  fluid  with  the 


24  MANUAL  OF  BACTERIOLOGY. 

low  power  and  then  to  focus  upon  this  edge  with  the  oil-im- 
mersion objective.  Above  all  things  guard  against  breaking 
the  cover-glass  by  forcing  the  objective  down  upon  it.  The 
motility  of  certain  bacteria  is  one  of  the  most  striking  phenom- 
ena to  be  observed  in  the  hanging-drop.  It  is  not  to  be  con- 
fused with  the  so-called  "Brownian  movement"  which  is  ex- 
hibited by  fine  particles  suspended  in  a  watery  fluid.  It  is  well 
for  the  beginner  to  observe  the  character  of  the  Brownian  move- 
ment by  rubbing  up  some  dry,  powdered  carmine  in  a  little 
water,  and  with  the  microscope  to  study  the  trembling  motion 
exhibited  by  these  particles  of  carmine.  It  will  be  noticed 
that,  although  the  particles  oscillate,  no  progress  in  any  direc- 
tion is  accomplished  unless  there  are  currents  in  the  fluid. 
Such  currents  might  give  rise  to  the  impression  that  certain 
bacteria  possessed  motility  when  they  were,  in  fact,  powerless 
to  move  of  themselves.  In  the  hanging-drop  the  multiplica- 
tion of  bacteria  can  be  studied,  the  formation  of  spores  and  the 
development  of  spores  into  fully  formed  bacteria.  The 
hanging-drop  has  recently  been  put  into  service  for  the  demon- 
stration of  the  so-called  serum-reaction  with  the  bacillus  of 
typhoid  fever.  Sometimes  bacteria  must  be  watched  in  the 
hanging-drop  for  hours,  or  even  days,  and  it  may  be  necessary 
to  keep  it  at  the  temperature  of  the  human  body  for  this  length 
of  time.  Various  complicated  kinds  of  apparatus  have  been 
devised  for  this  purpose,  but  they  are  needful  only  with  special 
kinds  of  work.  When  the  hanging-drop  preparation  is  no 
longer  required,  the  slide  and  cover-glass  should  be  dropped 
into  a  5  per  cent,  carbolic  acid  solution  and  afterward  sterilized 
by  steam. 

Hanging  block  preparations,  which  were  introduced  by 
Hill,*  consist  in  the  use  of  a  cube  of  nutrient  agar  instead  of  a 
drop  of  fluid.  Bacteria  are  distributed  on  the  surface  of  the 
agar,  which  is  then  applied  to  a  cover-glass,  and  mounted  like 

*  Journal  of  Medical  Research.     Vol.  VII.     March,  1902. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         25 

a  hanging-drop.  The  bacteria  are  kept  in  a  layer  close  to  the 
glass,  where  growth  may  be  studied. 

Cover- glass  Preparations. — The  study  of  bacteria  with 
the  microscope  is  for  the  most  part  done  by  means  of  smears 
made  upon  thin  cover-glasses.  It  is  best  to  obtain  the  kind 
sold  by  dealers  as  No.  i,  J-inch  squares. 

The  cover-glass  may  be  cleaned  best  by  immersion  in  a 
mixture  of  sulphuric  acid  and  bichromate  of  potassium  solu- 
tion, and  afterward  washed  thoroughly  in  distilled  water,  and 
finally  in  alcohol.  A  stock  of  clean  cover-glasses  may  be  kept 

CLEANING  FLUID. 

Potassium  bichromate 40  grams. 

Water .   150  c.c. 

Dissolve  the  bichromate  of  potassium  in  the  water,  with 
heat;  allow  it  to  cool;  then  add  slowly  and  with  care 
sulphuric  acid,  commercial 230  c.c. 

in  a  bottle  of  alcohol,  or  perhaps  preferably  in  alcohol  contain- 
ing 3  per  cent,  of  hydrochloric  acid.  When  they  are  needed  for 
use  they  should  be  wiped  clean  with  a  piece  of  linen  cloth.  If 
they  are  heated  at  the  time  the  preparations  are  made  the 
bacteria  will  be  found  to  spread  more  readily  than  on  the  cold 
surface  and  to  adhere  better.  Whenever  it  is  taken  into  the 
fingers  it  should  be  held  by  the  edges,  never  by  the  flat  surfaces. 
In  spreading  bacteria  upon  it  and  in  all  subsequent  manipula- 
tions, as  staining,  the  cover-glass  should  be  handled  with  the 
forceps.  It  can  be  used  very  conveniently  in  the  form  of  for- 
ceps known  as  the  Cornet  forceps,  or  in  the  modification  de- 
vised by  Stewart.  Bacteria  may  be  placed  upon  the  cover- 
glass  by  allowing  the  glass  to  fall  upon  one  of  the  colonies  of 
bacteria,  on  a  gelatin  or  agar  plate  (see  page  98),  which  will 
adhere  to  it  in  part,  producing  an  "impression  preparation" 
(German,  Klatschpreparat).  Such  a  preparation,  after  drying 
in  the  air,  is  to  be  fixed  by  passing  it  through  the  flame  three 


26 


MANUAL  OF  BACTERIOLOGY. 


times.     (See  below.)     The  forceps  with  which  it  is  handled 
should  be  sterilized  in  the  flame. 

Generally  bacteria  contained  in  fluids,  like  sputum,  or  taken 
from  the  surface  of  a  culture,  are  smeared  over  the  cover-glass 
by  means  of  the  platinum  wire  or  loop,  which  must  be  heated 
to  a  red  heat  before  and  after  the  operation.  Such  prepara- 


FIG.  7.— Cornet  forceps  for  cover-glasses. 

tions  are  called  smear,  cover-glass,  cover-slip,  or  film  prep- 
arations. When  the  material  to  be  spread  is  thick  or  very 
viscid,  a  small  drop  of  distilled  water  must  first  be  placed  in  the 
center  of  the  cover-glass  so  as  to  dilute  it.  Beginners  generally 
take  too  much  material  on  the  wire.  As  thin  a  smear  as 
possible  is  made.  It  is  allowed  to  dry  in  the  air;  this  should 
occupy  a  few  seconds.  The  drying  may  be  hastened  by  hold- 


FIG.  8.— Stewart  forceps  for  cover-glasses. 


ing  the  forceps  with  the  cover-glass  a  long  distance  above  the 
flame,  at  a  point  where  the  heat  would  cause  no  discomfort 
to  the  hand.  Having  dried  the  preparation,  it  is  to  be  passed 
with  the  smeared  surface  up  three  times  through  the  flame  of 
a  Bunsen  burner  or  alcohol  lamp.  This  should  not  be  done  too 
slowly  and  yet  sufficiently  so  to  fix  the  preparation.  Various 
directions  are  given  by  different  authors  as  to  the  time  which 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         27 

should  be  employed,  but  none  of  these  appear  exact,  and  one 
soon  acquires  by  practice  an  idea  as  to  how  long  the  manip- 
ulation should  take.  Indeed,  this  varies  with  the  character 
of  the  preparation.  The  heat  of  the  flame  serves  to  dry  the 
bacteria  upon  the  cover-glass  and  make  them  adhere  per- 
manently in  position.  It  also  aids  in  the  penetration  of  the 
staining  dyes;  but  it  is  not  sufficient,  when  applied  in  this 
manner,  to  kill  all  kinds  of  bacteria,  especially  those  containing 
spores.  After  it  has  been  passed  through  the  flame  three  times, 
the  preparation  may  be  stained  with  a  solution  of  one  of  the 
aniline  dyes,  as  described  below,  and  after  washing  in  water 
and  drying  may  be  mounted,  face  down,  in  Canada  balsam 


FIG.  9. — Kirkbride  forceps  for  holding  slides. 


upon  a  glass  slide.     It  makes  a  suitable  object  to  be  examined 
with  the  oil-immersion  objective. 

The  smear  preparation  may  equally  well  be  made  directly 
upon  the  glass  slide.  The  fixation  in  the  flame  must  then 
occupy  a  longer  time  than  with  the  small  and  thin  cover-glass. 
Such  preparations  have  the  advantage  that  several  may  be 
made  upon  one  slide,  and  that  after  staining  them  they  may  be 
examined  in  cedar-oil,  with  the  oil-immersion  lens,  without 
the  use  of  the  cover-glass  and  Canada  balsam.  The  forceps  of 
Kirkbride  will  be  found  convenient  for  staining  on  the  slide. 
Experiments  performed  in  the  writer's  laboratory  have  shown 
that  the  ordinary  method  of  fixation  in  the  flame,  when  ap- 
plied to  bacteria  spread  upon  slides,  has  little  effect  on  the 


28  MANUAL  OF  BACTERIOLOGY. 

vitality  of  many  species.  The  beginner  is,  therefore,  advised 
to  make  his  preparations  on  cover-glasses. 

When  very  resistant  or  dangerous  pathogenic  bacteria  are 
being  handled,  after  fixation  by  heat  upon  the  slide  or  cover- 
glass,  the  preparation  may,  if  desired,  be  immersed  in  i-iooo 
solution  of  bichloride  of  mercury  long  enough  to  kill  the 
bacteria,  without  injuring  the  preparation  or  interfering  with 
its  staining  properties. 

Staining. — The  bacterial  cells  are  devoid  of  color,  and  the 
object  of  staining  them  is  to  give  them  artificially  some  color 
that  would  make  them  distinct  and  easily  visible  with  the  micro- 
scope. In  order  that  they  shall  stand  out  sharply  the  stain 
employed  should  leave  the  background  unstained.  This 
result  is  best  obtained  with  aqueous  solutions  of  the  aniline 
dyes.  These  aniline  dyes,  so  called,  are  derivatives  of  coal-tar, 
but  not  always  of  aniline.  They  are  indispensable  in  bacterio- 
logical work.  Their  number  is  very  large,  but  only  a  few  are  in 
common  use.  It  is  important  to  have  the  purest,  and  those 
manufactured  by  Griibler  are  reliable. 

It  is  simplest  to  classify  the  aniline  dyes  as  acid  or  basic. 
Eosin,  picric  acid  and  acid  fuchsin  are  acid  dyes;  they  tend  to 
stain  tissues  diffusely.  Fuchsin,  gentian-violet  and  methy- 
lene-blueare  basic  dyes;  they  have  an  affinity  for  the  nuclei  of 
tissues  and  for  bacteria;  they  therefore  are  the  dyes  used  chiefly 
in  bacteriological  work.  The  other  kinds  may  be  employed 
as  contrast-stains;  another  contrast-stain  frequently  used  is 
Bismarck  brown.  It  is  best  to  keep  on  hand  saturated  solu- 
tions of  the  aniline  dyes  in  alcohol,  from  which  watery  solu- 
tions may  be  made  when  needed  by  adding  a  few  drops  of  the 
alcoholic  solution  to  a  small  dish  filled  with  water.  The 
alcoholic  solution  is  diluted  about  ten  times,  so  as  to  make  a 
liquid  which  is  just  transparent  in  a  layer  about  12  mm.  in 
thickness,  after  filtering. 

Fuchsin  and  gentian -violet  operate  rapidly  and  intensely. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         29 

Methylene-blue  works  more  slowly  and  feebly;  it  is  to  be  pre- 
ferred where  the  bacteria  occur  in  thick  or  viscid  substances, 
like  pus,  mucus  and  milk,  and  acts  more  energetically  when 
made  slightly  alkaline. 

Method  of  Staining  Cover-glass  Preparations.— (a)  A 
smear  preparation  of  bacteria  having  been  made,  dried  and 
passed  through  the  flame  three  times  in  the  manner  above 
described,  and  a  watery  solution  of  either  fuchsin,  gentian- 
violet  or  methylene-blue  having  been  prepared,  the  cover-glass 
is  to  be  dropped  into  a  dish  containing  the  dye,  or  the  dye  may 
be  dropped  upon  the  cover-glass  held  in  the  forceps. 

(b)  Allow  the  stain  to  act  for  about  thirty  seconds. 

(c)  Wash  in  water. 

(d)  Examine  with  the  microscope  in  water  directly  or  after 
drying  and  mounting  in  Canada  balsam. 

The  rapidity  and  intensity  of  staining  may  be  increased  by 
warming  the  solution  slightly.  The  preparation  may  also  be 
improved  by  rinsing  for  a  few  seconds  in  \  per  cent,  acetic  acid 
after  staining.  If  this  is  done  quickly,  and  the  preparation 
thoroughly  washed  in  water  it  does  not  materially  affect  the 
stain  in  the  bacteria,  and  it  clears  up  the  background,  thus 
bringing  out  the  bacteria  in  stronger  relief.  The  acid  should 
be  merely  poured  on  and- poured  off,  otherwise  the  bacteria  will 
be  more  or  less  decolorized. 

Preparations  that  are  mounted  at  first  in  water  may  be  made 
permanent  by  letting  a  drop  of  water  fall  at  the  edge  of  the 
cover-glass  so  that  it  may  easily  be  removed  from  the  slide,  then 
drying  and  mounting  in  Canada  balsam.  Cover-glass  prep- 
arations which  have  been  stained  are  examined  with  oil- 
immersion  objective,  employing  the  plane  mirror,  having  the 
iris  diaphragm  open  and  the  condenser  close  to  the  lower  sur- 
face of  the  glass  slide.  The  purpose  is  to  obtain  the  most  in- 
tense illumination  possible  over  a  small  field.  The  watery 
solutions  of  aniline  dyes  prepared  as  above  described  deterior- 


30  MANUAL  OF  BACTERIOLOGY. 

ate  in  a  short  time,  and  it  is  best  to  prepare  them  freshly  each 
time  they  are  required.  A  very  useful  solution,  which  is 
permanent,  is  Loffler's  alkaline  methylene-blue: 

Concentrated  alcoholic  solution  of  methylene-blue    .    .    .     30  c.c. 
Potassium  hydrate  (caustic  potash),   1-10,000  watery 

solution 100  c.c. 

Loffler's  methylene-blue  is  a  good  stain  for  general  purposes. 
It  is  perhaps  more  in  use  than  any  other  formula  for  coloring 
the  diphtheria  bacillus. 

Aniline-water  Staining  Solutions. — The  intensity  with 
which  aniline  dyes  operate  may  be  increased  by  adding  aniline 
oil  to  the  solution : 

Aniline  oil 5  c.c. 

Water       100  c.c. 

Mix,  shake  vigorously,  filter;  the  fluid  after  filtration  should 
be  perfectly  clear;  add— 

Alcoholic  solution  of  fuchsin  (or  gentian-violet,  or  methy- 
lene-blue)      ii  c.c. 

Alcohol 10  c.c. 

Aniline-water  staining  solutions  do  not  keep  well,  and  need 
to  be  freshly  prepared  about  every  ten  days  or  two  weeks. 
The  keeping  quality  depends  probably  upon  the  temperature 
and  possibly  on  the  exposure  to  light.  Sometimes  it  keeps 
longer  than  at  others,  even  when  prepared  each  time  alike.  It 
is  a  good  plan  to  filter  it  every  time  before  use.  Precipitates 
form  during  the  first  twenty-four  hours  after  the  stain  is  made, 
and  for  this  reason  cleaner  preparations  are  obtained  with  the 
stain  after  it  has  stood  for  a  day.  The  alcohol  advised  in  the 
accompanying  formula  tends  to  dissolve  the  precipitate.  Vari- 
ous other  formulae  are  given  by  different  authors  for  the  prep- 
aration of  aniline-water  staining  solutions,  but  the  one  given 
above  will  be  found  to  give  satisfactory  results.  The  appli- 
cations of  the  aniline-water  stains  will  be  given  under  separate 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         31 

headings.     In  general,  however,  they  are  employed  where  a 
stain  of  unusual  power  is  required. 

Gram's  Method. — The  advantages  .of  Gram's  method  are 
that  by  using  it  certain  kinds  of  bacteria  may  be  stained  a  violet 
color,  while  other  bacteria  are  stained  feebly  or  not  at  all. 
Cover-glass  preparations,  having  been  prepared  preferably 
from  agar  slant  cultures  twelve  to  twenty-four  hours  old* 
and  fixed  in  the  usual  manner  (see  pages  26  and  27),  are 
stained  as  follows.  Some  advise  short  rinsing  with  water 
after  pouring  off  the  staining  solution. 

(a)  Stain  in  aniline- water  gentian-violet  solution,  from  two 
to  five  minutes.     The  intensity  of  the  stain  may  be  increased 
by  warming  slightly. 

(b)  Iodine  solution,  one  and  one-half  minutes: 

Iodine i  gram. 

Potassium  iodide 2  grams. 

Water 300  c.c. 

In  this  solution  the  preparation  becomes  nearly  black. 

(c)  Wash    repeatedly    in    strong,    preferably    in    absolute 
alcohol;  the  alcohol  becomes  stained  with  clouds  of  violet 
coloring  matter;  the  alcohol  is  used  as  long  as  the  violet  color 
continues  to  come  away,  and  until  the  preparation  is  decolor- 
ized or  has  only  a  faint  gray  tint.    It  may  be  necessary  to  repeat 
the  treatment  with  the  iodine  solution. 

(d)  When  desired,  the  specimens  may  be  stained,  by  way  of 
contrast,  with  a  watery  solution  of  Bismarck  brown  or  eosin. 

(e)  Wash  in  water,  and  examine  either  in  water  ^directly  or 
after    drying    and    mounting    in    Canada    balsam.     Gram's 
method  and  its  modifications  are  not  always  trustworthy  for 
diagnostic  purposes;  since  one  and  the  same  organism  may 
stain  but  faintly  if  all  at  one  time,  and  quite  intensely  at 
others.!     Still  it  is  a  more  or  less  useful  aid,  particularly  with 

*Muir  and  Ritchie.  American  Ed.,  1903,  p.  103.  Also  Mallory  and  Wright. 
1904,  p.  101. 

fKolle  and  Wassermann.     Vol.  I,    p.  70. 


32  MANUAL  OF  BACTERIOLOGY. 

practice.     Bearing  this  qualification   in   mind,  the  following 
lists  will  serve  as  a  guide. 

Bacteria  that  are  stained  by  Gram's  method : 

Staphylococcus  pyogenes  aureus, 

Streptococcus  pyogenes, 

Micrococcus  lanceolatus  (of  pneumonia), 

Micrococcus  tetragenus, 

Bacillus  of  diphtheria, 

Bacillus  of  tuberculosis, 

Bacillus  of  leprosy, 

Bacillus  of  anthrax, 

Bacillus  of  tetanus, 

Bacillus  aerogenes  capsulatus, 

Ray  fungus  of  actino mycosis. 

Of  these  the  tubercle  bacillus  and  the  bacillus  of  leprosy 
require  a  much  longer  exposure  to  the  stain  than  other  bac- 
teria in  the  list. 

List  of  bacteria  that  are  not  stained  by  Gram's  method : 

Gonococcus, 

Diplococcus  intracellularis  meningitidis, 

Micrococcus  melitensis, 

Bacillus  of  chancroids  (Ducrey), 

Bacillus  of  dysentery  (Shiga), 

Bacillus  of  typhoid  fever, 

Bacillus  coli  communis, 

Bacillus  pyocyaneus, 

Bacillus  of  influenza, 

Bacillus  of  bubonic  plague, 

Bacillus  of  glanders  (bacillus  mallei), 

Bacillus  of  Friedlander, 

Bacillus  proteus, 

Spirillum  of  Asiatic  cholera, 

Spirillum  of  relapsing  fever. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        33 

Staining  the  Bacillus  of  Tuberculosis. — Since  the  tu- 
bercle bacillus  does  not  take  the  ordinary  stains  readily,  a  very 
large  number  of  methods  have  been  proposed  for  staining  it, 
all  of  which  depend  upon  the  principle  that,  after  adding  to 
solutions  of  aniline  dyes  certain  substances,  like  aniline-water, 
carbolic  acid,  or  solutions  of  ammonia  or  soda,  the  bacillus 
tuberculosis  is  stained  with  great  intensity,  and  gives  up  its 
stain  with  difficulty.  Solutions  of  acids  will  remove  the  stain 
from  all  parts  of  the  preparation  excepting  from  the  tubercle 
bacilli,  which  retain  the  dye,  once  having  acquired  it.  The 
rest  of  the  preparation  may  now  be  given  a  different  color— 
contrast-stain. 

Bacilli  that  resist  decolorization  by  acids  are  called  acid- 
proof  or  acid-fast.  The  most  important  are  tubercle  and 
leprosy  bacilli.  There  are  various  other  species,  however,  most 
of  which  are  less  resistant  to  acids  and  alcohol  than  tubercle 
bacilli.  They  are  discussed  in  the  article  on  the  bacillus 
tuberculosis  in  Part  IV. 

Occasionally  spores  of  other  bacteria,  micrococci  and  horny 
epithelial  cells  are  imperfectly  decolorized,  but  their  forms  dis- 
tinguish them  from  tubercle  bacilli.  Minute  crystalline 
needles  which  have  a  shape  like  that  of  bacilli  are  often  en- 
countered in  sputum,  but  their  nature  will  be  recognized  after 
a  little  practice. 

The  stain  for  tubercle  bacilli  is  most  frequently  used  for 
specimens  of  sputum  from  cases  o  f  suspected  pulmonary  tu- 
berculosis; it  may  be  applied  to  other  fluids  and  secretions 
equally  well.  It  is  not  reliable,  however,  when  applied  to 
milk,  as  the  oil  present  in  milk  interferes  with  its  operation, 
and  milk  and  its  products  quite  often  contain  other  acid-proof 
bacilli.  The  smegma  of  the  external  genitals  also  frequently 
contains  acid-proof  bacilli  that  are  not  tubercle  bacilli.  On 
this  account  all  fluids  and  discharges  from  the  genito-urinary 
tract  need  to  be  examined  with  particular  care  not  to  confuse 


34  MANUAL  OF  BACTERIOLOGY. 

tubercle  bacilli  with  smegma  bacilli.  (See  smegma  bacilli  in 
Part  II.,  Chapter  IV.) 

Patients  should  be  given  minute  instructions  concerning  the 
collection  of  sputum.  The  bottle  used  should  be  new,  wide- 
mouthed,  clean,  and  kept  tightly  stoppered  with  a  clean  cork. 
The  patient  should  be  cautioned  against  allowing  the  expec- 
toration to  get  on  the  outside  of  the  bottle.  Probably  what- 
ever risk  is  incurred  by  those  who  examine  sputum  comes 
chiefly  from  the  outside  of  the  bottle  having  been  soiled  with 
sputum  containing  tubercle  bacilli.  Often  little  white  particles 
may  be  seen  floating  in  the  mucous  portions  of  the  sputum. 
These  particles  should  be  selected  for  the  investigation,  and 
may  be  spread  in  a  thin  film  on  the  cover-glass  with  the  plati- 
num wire,  which  is  sterilized  in  the  flame  before  and  after 
using.  The  selection  of  the  little  white  particles  will  be  facili- 
tated if  the  sputum  be  poured  into  a  clean  glass  dish,  which 
may  be  placed  on  a  black  surface.  A  form  of  porcelain  dish  is 
furnished  by  dealers,  the  bottom  of  which  is  black,  and  which 
is  convenient,  for  these  manipulations.  The  smears  must  be 
made  thin,  or  the  subsequent  decolorization,  after  staining,  will 
not  be  uniform.  It  is  hardly  necessary  to  observe  that  the 
operator  must  be  scrupulously  careful  not  to  contaminate  the 
material  under  examination  with  any  kind  of  extraneous 
matter.  The  cover-glasses  and  slides  which  are  used  should  be 
new,  and  should  have  been  cleaned  with  bichromate  of  potas- 
sium and  sulphuric  acid  (see  page  25). 

When  the  work  is  completed,  the  bottle  containing  the 
sputum  should  be  sterilized  by  steam  or  boiling. 

Many  different  methods  for  staining  the  tubercle  bacillus 
have  been  proposed.  In  most  of  those  now  in  use  the  following 
solution  (Ziehl's  carbol-fuchsin)  is  employed— 

Fuchsin i  gram. 

Carbolic  acid,  pure 5  c.c. 

Alcohol 10  c.c. 

Distilled  water 100  c.c. 

N.  B. — Acid  fuchsin  cannot  be  used  for  this  stain. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        35 

The  method  given  below  is  the  one  recommended. 
Method  for  staining  the  tubercle  bacillus : 

(a)  The  cover-glass  preparation  is  made,  dried  and  fixed  by 
passing  through  the  flames  three  times  (see  pages  26  and  27). 

(b)  The  cover-glass,  held  in  forceps  or  in  a  watch-crystal,  is 
covered  with  carbol-fuchsin  and  heated  till  bubbles  begin  to 
appear  showing  that  the  water  in  the  stain  is  boiling.     The 
stain  should  be  allowed  to  act  for- five  minutes  and  kept  hot 
during  this  time.     It  is  not  always  necessary  to  heat  as  high  as 
this  nor  to  allow  the  stain  to  remain  for  so  long  a  time;  but  in 
order  to  be  sure  in  all  cases  it  is  best  to  do  so. 

(c)  Wash  in  water. 

(d)  Wash  in  alcohol  containing  3  per  cent,  of  hydrochloric 
acid  one  minute,  or  longer  if  necessary  to  remove  the  red  color. 

(e)  Wash  in  water. 

(/)  Stain  with  methylene-blue  solution  (see  page  30)  thirty 
seconds. 

(g)  Wash  in  water. 

It  may  be  found  necessary  to  repeat  the  treatment  with 
acid  alcohol,  and  this  should  be  done  if  the  color  returns  to  the 
preparation  after  washing  in  water. 

(h)  Examine  in  water  directly,  or  after  drying  and  mounting 
in  Canada  balsam.  'Tubercle  bacilli  take  a  brilliant  red 
color;  other  bacteria  and  the  nuclei  of  cells  are  stained 
blue. 

Gabbett's  Method. — This  method  is  very  popular  and  widely 
used  on  account  of  its  convenience.  It  is  not  as  reliable  as  the 
one  just  given. 

Gabbett's  solution : 

Methylene-blue i  to  2  grams. 

25  per  cent,  watery  solution  of  sulphuric  acid 100  c.c. 

(a)  The  cover-glass  preparation  is  to  be  made,  dried  and 
fixed  by  passing  through  the  flame  three  times. 


36  MANUAL  OF  BACTERIOLOGY. 

(b)  The  carbol-fuchsin  stain  is  applied  from  two  to  five 
minutes  to  the  cover-glass,  held  in  forceps  or  in  a  watch-crys- 
tal; it  need  not  be  warmed. 

(c)  Wash  in  water. 

(d)  Gabbett's  solution  is  applied  for  one  minute. 

(e)  Wash  in  water.     The  preparation  should  have  a  blu^ 
color.     It  may  be  examined  in  water  directly  or  after  drying 
and  mounting  in  Canada  balsam. 

Gabbett's  method  has  the  advantage  of  decolorizing  the 
preparation  and  staining  the  background  with  methylene-blue 
at  the  same  time.  Tubercle  bacilli  are  colored  a  brilliant  red; 
most  other  bacteria  and  the  nuclei  of  cells  are  colored  blue. 
The  acid-proof  bacilli  mentioned  on  page  33  also  retain  the  red 
stain  in  most  cases,  and  might  be  confused  with  tubercle 
bacilli. 

Of  the  numerous  methods  of  staining  tubercle  bacilli,  only 
a  few  others  can  be  mentioned.  Aniline-water  fuchsin,  aniline- 
water  gentian-violet  or  carbol-fuchsin  may  be  used.  The 
intensity  of  the  stain  may  then  be  increased  by  warming  the 
preparation  till  it  steams  or  boils,  and  allowing  the  warm  stain 
to  act  on  the  specimens  for  from  three  to  five  minutes;  the  prep- 
aration may  also  be  left  in  the  cold  stain  over  night.  De- 
colorization  of  the  background  may  be  effected  with  a  25  per 
cent,  solution  of  sulphuric  acid  used  till  the  color  disap- 
pears, or  a  30  per  cent,  solution  of  nitric  acid,  which 
operates  very  rapidly.  If  the  color  persists  after  wash- 
ing in  water,  it  should  be  dipped  in  the  acid  again.  After 
either  acid  the  preparation  is  to  be  washed  in  alcohol  until 
the  last  trace  of  the  stain  has  been  removed.  '  An  excellent 
decolorizing  agent  is  a  3  per  cent,  solution  of  hydrochloric  acid 
in  alcohol,  used  for  about  a  minute.  With  any  of  these  acid 
solutions  the  decolorization  can  be  accomplished  more  per- 
fectly than  with  Gabbett's  solution,  where  the  operation  of  the 
decolorizing  agent  is  masked.  The  contrast-stain  may  be 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        37 

(emitted  entirely  if  it  is  desired.  A  suitable  contrast-stain 
liter  fuchsin  staining  is  a  solution  of  methylene-blue;  after 
gentian-violet  staining,  Bismarck  brown. 

Those  who  have  had  experience  in  staining  tubercle  bacilli 
jjoon  discover  that  the  bacilli  exhibit  some  differences  in  their 
Resisting  power  to  strong  acids.  One  encounters  occasionally 
bacilli  that  are  perfectly  stained  side  by  side  with  others  that 
Ire  more  or  less  completely  decolorized.  These  facts  show  the 
necessity  of  practice  with  any  method,  and  of  exercising  caution 
Lnd  judgment  in  making  a  diagnosis  where  the  number  of 
bacilli  happens  to  be  scanty.  If  tubercle  bacilli  are  not  found 
n  the  first  preparation,  other  preparations  should  be  made, 
bometimes  a  large  number  of  cover-glasses  must  be  examined. 

Various  expedients  have  been  devised  to  concentrate  tubercle 
bacilli  when  only  a  small  number  may  be  present  in  a  sample 
tf  sputum.  In  Biedert's  method  about  15  c.c.  of  sputum  are 
mixed  with  5  c.c.  of  distilled  water,  4  to  8  drops  of  sodium 
hydrate  solution  are  added,  and  the  mixture  is  boiled.  After 
boiling,  about  15  c.c.  of  distilled  water  are  added.  The 
mixture  may  be  set  aside  in  a  conical  glass  for  from  twenty- 
lour  to  forty-eight  hours  to  allow  sedimentation  or  the  sedi- 
pient  may  be  precipitated  rapidly  by  the  use  of  the  centrifuge. 
In  either  case  cover-glass  preparations  are  made  from  the 
pediment  in  the  manner  already  described.  The  sediment 
[will  be  found  to  have  little  adhesive  power,  and  will  not  stick 
Swell  to  the  cover-glass.  It  is  convenient  to  save  some  of  the 
original  sputum  and  mix  it  with  the  sediment  for  this  purpose. 

Fixation  of  Tissues.— Pieces  of  organs  about  i  cm.  in 
thickness  may  be  taken.  Alcohol  is  the  best  agent  for  pre- 
serving them.  The  fixation  will  be  completed  in  a  few 
jdays.  It  is  best  to  change  the  alcohol.  The  amount  of  the 
•.alcohol  must  be  twenty  times  the  bulk  of  the  tisuse  to  be  pre- 
served. 

Ten  parts  of  the  standard  40  per  cent,  solution  of  formalde- 


38  MANUAL  OF  BACTERIOLOGY. 

hyde,  with  90  parts  water  make  a  good  mixture  for  fixation; 
after  twenty-four  hours  change  to  alcohol. 

Imbedding  in  Collodion  or  Celloidin.— From  alcohol  the 
pieces  of  tissue  are  placed  in  equal  parts  of  alcohol  and  ether, 
twenty-four  hours;  thin  collodion  (ij  per  cent.),  twenty-four 
hours;  thick  collodion  of  a  syrupy  consistency  (6  per  cent.), 
twenty-four  hours.  The  specimen  is  laid  upon  a  block  of 
wood  or,  better,  the  compressed  vegetable  fibre  called  vul- 
canite, and  surrounded  by  thick  collodion,  and  then  placed 
in  70  per  cent,  alcohol.  The  collodion  makes  a  firm  mass, 
surrounding  and  permeating  the  tissue,  and  permits  very  thin 
sections  to  be  cut.  The  soluble  cotton  sold  by  dealers  in 
photographers'  supplies  serves  as  well  as  the  expensive  prep- 
aration known  as  celloidin.  To  make  collodion,  dissolve  it 
in  equal  parts  of  alcohol  and  ether.  Soluble  cotton  is  also 
called  pyroxylin,  and  is  a  kind  of  gun-cotton. 

Imbedding  in  Paraffin. — Pieces  of  tissue  2  to  3  mm. 
thick  which  have  already  been  fixed  in  alcohol  or  formalde- 
hyde are  to  be  placed : 

(a)  In  absolute  alcohol  for  twenty-four  hours. 

(b)  In  pure  xylol  one  to  three  hours. 

(c)  In  a  saturated  solution  of  paraffin  in  xylol  one  to  three 
hours. 

(d)  In  melted  paraffin  having  a  melting-point  of  50°  C., 
which  requires  the  use  of  a  water-bath  or  oven,  one  to  three 
hours.     The  xylol  must  be  entirely  driven  off,  and  the  tissue 
thoroughly  infiltrated. 

(e)  In  fresh  paraffin  for  one  hour. 

The  tissue  is  finally  placed  in  a  small  dish  or  paper  box 
and  covered  with  the  melted  paraffin.  The  paraffin  should 
be  hardened  as  quickly  as  possible  with  running  water.  It 
is  important  to  fix  the  piece  of  tissue  in  fhe  desired  position 
before  pouring  in  the  melted  paraffin. 

Paraffin  imbedding  is  especially  useful  when  serial  sections 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         39 

re  to  be  made.     Sections  of  exquisite  thinness  are  possible 
rith  this  form  of  imbedding.     The  knife  need  not  be  wet. 
In  order  to  mount  the  sections,  proceed  as  follows: 
(a)  Place   the   sections  on   water  in   a  porcelain   capsule. 
Varm  slightly,  when  the  sections  will  flatten  nicely.     Smear 
le  surface  of  a  slide  with  a  very  thin  layer  of  Mayer's  glycerin- 
[bumen  mixture.     Dip  the  slide  under  the  sections;  raise  it  up, 


FIG.  10. — Schanze  Microtome. 


and  drain  off  the  water,  leaving  the  sections  adhering  to  the 
glass  in  their  proper  positions.  Let  them  dry  for  some  hours 
in  the  incubator,  and  they  will  be  firmly  fastened  to  the  slide. 

(b)  Dissolve  out  the  paraffin  in  one  of  the  numerous  solvents 
(xylol, 'a  few  minutes). 

(c)  Wash  off  the  xylol  with  absolute  alcohol,  and 

(d)  Stain  the  sections  as  desired. 

(e)  Dehydrate  in  absolute  alcohol. 


40  MANUAL  OF  BACTERIOLOGY. 

(/)   Clear  in  xylol. 
(g)  Mount  in  balsam. 

GLYCERIN-ALBUMEN  MIXTURE  (MAYER). 

Equal  parts  of  white  of  egg  and  glycerin  are  thoroughly  mixed,  and  then 
filtered.  'Add  a  little  gum-camphor  to  preserve. 

Section  Cutting. — Cutting  is  best  done  with  an  instrument 
called  a  microtome.  The  tissues  may  be  imbedded  in  collo- 
dion or  paraffin;  or  when  they  have  been  hardened  with  for- 
maldehyde they  may  be  cut  after  freezing.  Bacteria  stain  ad- 
mirably in  such  frozen  sections.  For  routine  work  collodion 
imbedding  will  be  found  as  convenient  a  process  as  any. 
Paraffin  imbedding  gives  the  thinnest  sections. 

A  microtome  consists  of  a  heavy,  sliding  knife-carrier, 
which  moves  with  great  precision  on  a  level,  and  of  a  device  for 
elevating  the  object  which  is  to  be  cut  any  desired  distance 
after  each  excursion  of  the  knife.  The  thickness  of  the  section 
will  be  the  distance  which  the  object  is  elevated.  The  knife  is 
kept  wet  with  alcohol  during  the  cutting  of  collodion  sections, 
otherwise  it  is  left  dry.  The  microtome  is  usually  provided 
with  a  special  form  of  knife.  A  razor  will  serve  nearly  as  well, 
after  having  had  the  lower  side  ground  flat.  If  a  razor  is  used, 
a  special  form  of  razor-holder  must  be  attached  to  the  micro- 
tome to  receive  the  razor.  Above  all,  it  is  necessary  that  the 
knives  should  be  kept  in  good  condition.  Only  occasionally 
will  they  need  honing,  using  a  fine  water-stone  or  Belgian  hone. 
Preferably  the  knife  should  not  be  honed  directly  on  the  stone 
itself  but  on  a  piece  of  clean  plate  glass,  on  which  the  stone  is 
first  rubbed  with  water.  By  this  means  the  entire  cutting 
edge  is  sharpened  in  one  plane.  The  movement  in  honing 
should  be  from  heel  to  toe,  and  toward  the  cutting  edge,  always 
placing  the  back  of  the  knife  next  the  hone  when  turning.  The 
knife  should  be  stropped  frequently.  The  leather  of  the  strop 
should  be  glued  to  a  strip  of  wood  to  make  a  flat  surface.  The 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        41 

movement  in  stropping  should  be  from  toe  to  heel.  Sections 
should  be  cut  to  a  thickness  of  not  more  than  25  /*.  Thinner 
sections  (5  to  10  /*)  are  to  be  desired. 

Staining  of  Sections. — A  watery  solution  of  one  of  the 
basic  aniline  dyes  is  used — fuchsin,  gentian-violet  or  methylene- 
blue — made  by  adding  one  part  of  the  alcoholic  solution  to 
ten  parts  of  distilled  water.  Loffer's  solution  of  methylene- 
blue  serves  very  well. 

By  this  process  most  bacteria  are  stained;  also  the  nuclei 
of  cells;  frequently,  also,  certain  granules  contained  within 
some  cells  (German,  Mastzellen),  which  may  easily  be  mistaken 
for  bacteria  by  the  inexperienced  (basophilic  granules). 

(a)  Place  the  section  in  the  staining  solution  from  two  to 
five  minutes  or  longer. 

(b)  Wash  in  water. 

(c)  Place  in  a  watery  solution  of  acetic  acid,  ^  per  cent.,  for 
from  a  few  seconds  to  one  minute. 

(d)  Alcohol,  one  to  two  minutes;  change  to  absolute  alcohol. 
Touch  the  sections  to  blotting-paper  to  remove  the  superfluous 
alcohol. 

(e)  Xylol  until  clear;  xylol  is  to  be  preferred  to  othe<*  clearing 
agents,  like  oil  of  cloves,  most  of  which  slowly  remove  aniline 
colors.     It  has  the  disadvantage  of  not  clearing  when  the 
slightest  trace  of  water  is  present;  dehydration  in  alcohol  must, 
therefore,  be  complete.     The  section  should  be  removed  from 
the  xylol  as  soon  as  it  is  cleared;  otherwise  wrinkling  occurs. 

(/)  The  section  is  placed  upon  a  glass  slide;  a  drop  of 
Canada  balsam  is  placed  upon  it  and  then  a  cover-glass.  The 
Canada  balsam  should  be  dissolved  in  xylol. 

The  section  is  to  be  manipulated  with  straight  or  bent 
needles.  The  removal  from  xylol  to  the  glass  slide  is  managed 
best  with  a  spatula  or  section-lifter. 

The  above  statements  apply  to  frozen  sections  or  to  sections 
imbedded  in  celloidin.  Paraffin  sections  are  preferably  at- 


42  MANUAL  OF  BACTERIOLOGY. 

tached  to  the  slide  with  glycerin-albumen.  The  different  steps 
in  the  process  follow  in  the  same  order.  The  stain  may  be 
poured  on  the  slide,  or  the  slide  may  be  placed  in  a  large  dish 
full  of  staining  fluid.  (See  page  29.)  Celloidin  sections  may 
also  be  stained  on  the  slide.  If  the  section  be  well  spread  and 
flattened  thoroughly  with  blotting-paper,  it  will  usually  ad- 
here to  the  slide,  and  is  less  likely  to  wrinkle.  It  must  not  be 
allowed  to  dry. 

Gram's  Method  may  be  applied  to  the  staining  of  sections 
of  tissues  as  well  as  to  smears  upon  cover-glasses. 

(a)  Place  the  section  in   aniline-water  gentian-violet,   five 
minutes   or   longer.     See   the   preceding   paragraph   for   the 
manner  of  handling  sections. 

(b)  Rinse  in  water. 

(c)  Iodine  solution  (see  page  31),  one  and  one-half  minutes. 

(d)  Alcohol,  until  decolorized  to  a  faint  blue-gray. 

(e)  Xylol. 

(/)  Mount  on  a  slide  in  balsam. 

Weigert's  Modification  of  Gram's  Method. — (a)  Place 
the  section  in  aniline-water  gentian-violet  solution,  five  minutes 
or  more.  See  page  41  for  the  manner  of  handling  sections. 

(b)  Rinse  in  water. 

(c)  After   placing   the   section   upon   a   slide,    and   having 
straightened  it  carefully,  absorb  the  water  with  blotting-paper. 

(d)  Iodine  solution  (see  page  31),  one  to  two  minutes. 

(e)  Absorb  the  iodine  solution  with  blotting-paper. 

(/)  Add  an;line  oil,  removing  it  from  time  to  time  with  blot- 
ting-paper, and  adding  fresh  aniline  oil  until  the  specimen 
becomes  a  faint  blue-gray  and  until  the  color  ceases  to  come 
away.  (Aniline  oil  serves  in  this  connection  both  to  decolorize 
and  to  dehydrate.  It  absorbs  the  water  rapidly  and  efficiently. 
However,  on  account  of  its  decolorizing  tendency,  it  must  be 
removed  before  the  specimens  can  be  mounted  permanently.) 

(g)  Wash  in  several  changes  of  absolute  alcohol. 


.-      EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         43 

(h)  Add  xylol;  remove  it  with  blotting-paper;  and  add  fresh 
xylol  several  times,  in  order  to  extract  the  last  trace  of  aniline 
oil. 

(i)  Mount  in  Canada  balsam. 

This  method  is  more  convenient  for  the  staining  of  sections 
than  the  Gram  method.  The  results,  however,  are  essentially 
the  same  as  far  as  the  bacteria  are  concerned;  fibrin  is  usually 
stained  blue,  hyaline  material  is  also  stained  blue,  and  bacteria 
violet.  It  is  often  impossible  to  decolorize  the  nuclei  com- 
pletely without  decolorizing  the  bacteria  also.  The  parts  of 
the  nuclei  which  remain  stained  often  present  pictures  that 
resemble  bacteria,  and  which  may  lead  to  error  if  not  recog- 
nized. Basophilic  granules  also  retain  the  stain,  as  do  the 
horny  cells  of  the  epidermis.  These  remarks  apply  also  to 
Gram's  method,  except  as  regards  fibrin.  Very  beautiful 
preparations  can  be  obtained  according  to  this  or  the  Gram 
method  when  the  sections  have  previously  been  stained  in  car- 
mine; the  nuclei  will  then  be  colored  red,  bacteria  violet. 

Where  sections  are  stained  with  carmine  they  should  be 
thoroughly  washed  before  applying  the  Gram  stain,  since  the 
presence  of  acid  interferes  with  this  stain,*  and  any  of  the  acid 
alcohol  which  is  used  after  the  carmine  must  be  carefully  re- 
moved. 

Tubercle  bacilli  may  be  stained  in  sections  as  follows: 

(a)  Use  carbol-fuchsin,  or  aniline-water  gentian-violet  for 
one-half  to  two  hours  with  very  gentle  warming,  or  over  night 
without  warming.     See  page  41  for  the  manner  of  handling 
sections. 

(b)  Wash  in  water. 

(c)  Decolorize  with  some  one  of  the  decolorizing  agents 
mentioned  in  connection  with  the  staining  of  tubercle  bacilli  in 
cover-glass  preparations,  preferably  3  per  cent,  hydrochloric 
acid  alcohol.     Decolorization  must  be  continued  until  the  red 

*Kolle  and  Wassermann.     Vol.  I.,  p.  70. 


44  MANUAL  OF  BACTERIOLOGY. 

or  violet  color,  as  the  case  may  be,  has  disappeared,  which 
requires  one  to  several  minutes. 

(d)  Wash  thoroughly  in  alcohol. 

(e)  Wash  in  water. 

(/)  Use  hematoxylin  as  a  contrast-stain  for  fuchsin  prepara- 
tions, and  carmine  for  gentian-violet  preparations.  In  the 
latter  case  it  is  better  to  stain  with  carmine  before  staining 
the  bacilli.  The  carmine  is  not  affected  by  the  subsequent 
treatment. 

(g)  Wash  in  water. 

(h)  Alcohol. 

(i)  Xylol. 

(j)  Balsam. 

Tubercle  bacilli  may  also  be  stained  in  sections  by  Gram's 
method;  but  they  require  to  be  stained  in  the  gentian-violet 
aniline  oil  for  a  longer  time  than  do  other  bacteria  which  are 
stained  by  this  method.  The  same  statement  also  applies  to 
the  leprosy  bacilli  when  stained  by  Gram's  method. 

Nuclear  stains,  which  may  be  used  as  contrast-stains  for 
section : 

DELAFIELD'S  HEMATOXYLIN. 

Hematoxylin  crystals 4  grams. 

Alcohol 25  c.c. 

Ammonia  alum 50  grams. 

Water 400  c.c. 

Glycerin 100  c.c. 

Methyl-alcohol 100  c.c. 

Dissolve  the  hematoxylin  in  the  alcohol,  and  the  ammonia 
alum  in  the  water.  Mix  the  two  solutions.  Let  the  mixture 
stand  four  or  five  days  uncovered;  it  should  have  become  a 
deep  purple.  Filter  and  add  the  glycerin  and  the  methyl- 
alcohol.  After  it  has  become  dark  enough,  filter  again.  Keep 
it  a  month  or  longer  before  using;  the  solution  improves  with 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.         45 

age.     At  the  time  of  using,  filter  and  dilute  with  water  as 
desired. 

LITHIUM-CARMINE  ( O  RTH)  . 

Carmine 2.5  grams. 

Saturated  watery  solution  of  lithium  carbonate .    .    .    100.0  c.c. 

Bacteria  occasionally  stain  with  hematoxylin  though  as  a 
rule  but  faintly.  The  stain  is  not  used  for  bacteria  but  only 
for  staining  the  nuclei  of  the  tissue  cells. 

Add  .a  few  crystals  of  thymol.  The  carmine  dissolves 
readily  in  the  lithium  carbonate  solution.  Filter  the  stain  at 
the  time  of  using.  Sections  are  to  be  left  in  the  stain  five  to 
twenty  minutes. 

Sections  stained  in  carmine  are  placed  directly  in  acid 
alcohol  (i  part  hydrochloric  acid,  100  parts  70  per  cent,  alcohol) 
for  five  to  ten  minutes.  They  acquire  a  brilliant  scarlet  color. 
When  used  as  a  contrast-stain  for  tissues  containing  bacteria,  it 
is  best  to  use  it  before  staining  the  bacteria,  which  might  be 
decolorized  by  the  acid  alcohol.  The  sections  should  for  this 
reason  be  thoroughly  washed  before  applying  the  Gram  stain. 

Staining  of  Blood-films. — Romanowsky  Stain. — There  are 
various  modifications  of  this  stain.  MacNeal*  states  that  the 
following  formula  gives  the  best  result  for  physicians  and  for 
clinical  laboratories: 

Prepare  crude  methylene  violet  by  boiling  for  15  minutes 
over  a  free  flame  0.5  grams  of  methylene  azure,  0.5  grams  of 
sodium  carbonate  in  200  c.c.  of  distilled  water.  Cool  slightly 
and  filter.  The  crude  methylene-violet  separates  out  from  the 
filtrate  on  cooling.  Dry  the  crystals  thoroughly. 

Of  the  above  crude  methylene-violet 0.08  gr. 

Of  methylene-blue  (med.  pure) 0.08  gr. 

Of  eosin  (water  soluble  yellowish) 0.20  gr. 

Dissolve  in  100  c.c.  of  methyl  alcohol,  filter,  and  dilute  with 
10  c.c.  of  methyl  alcohol.  The  method  of  use  is  as  follows: 

*MacNeal.     Journ.  Infectious  Diseases.     Vol.  3.  1906,  pp.  412-433. 


46  MANUAL  OF  BACTERIOLOGY. 

Prepare  films  of  blood  as  directed  in  Chapter  VII.,  Part  I., 
and  allow  to  dry. 

(a)  Pour  the  stain  over  the  surface  of  the  preparation  till  it 
covers  it  and  allow  to  remain  for  one  miunte.    This  serves  to  fix 
the  film  of  blood  to  the  glass  as  well  as  to  stain  it,  so  it  is  not 
necessary  or  desirable  to  pass  the  preparation  through  the 
flame. 

(b)  Add  distilled  water,  drop  by  drop,  till  a  reddish  tint  ap- 
pears at  the  edges  and  a  metallic  scum  forms  on  the  surface.* 
About  six  drops  are  needed  for  a  three-fourths  inch  cover-glass. 
The  real  staining  of  the  preparation  now  takes  place,  and  re- 
quires two  or  three  minutes. 

(c)  Wash  in  distilled  water  till  the  thin  parts  of  the  prep- 
aration have  a  yellowish  or  pinkish  tint,  which  requires  one  to 
three  minutes. 

(d)  Dry  with  blotting-paper  and  mount  in  Canada  balsam. 
Bacteria,  malarial  parasities,  and  cell-nuclei  are  stained  blue, 

red  blood-corpuscles  are  orange-pink,  while  the  specific  granule 
of  the  leukocytes  (neutrophilic,  etc.)  appear  in  varoius  tints 
from  red  to  dark  blue.  The  chromatin  of  the  malarial  parasite 
takes  a  lilac  to  red  color.  The  blood-plates  have  a  bluish  or 
purplish  color  and  must  not  be  confused  with  malarial  parasites. 
Hastingsf  gives  directions  of  which  the  following  are  ap- 
parently the  essential  details:  Dissolve  2  grams  of  sodium 
carbonate  in  200  c.c.  hot  distilled  water  and  stir  in  2  grams 
methylene-blue  (Ehrlich  rectified).  Bring  to  a  boil  over  a  fire 
flame  or  boil  for  15  minutes  over  water  bath.  Replace  water 
lost  by  evaporation  and  heat  again  for  10  or  15  minutes. 
Pour  the  hot  solution  off  from  the  sediment  and  add  distilled 
water  up  to  200  c.c.  if  necessary.  Partially  neutralize  by  the 
addition  of  12.5  per  cent,  or  20  per  cent,  acetic  acid;  solution 
must  remain  alkaline.  Add  this  to  1000  c.c.  of  a  yV  per 

*Leishman.  British  Med.  Jour.,  1901,  II.,  757. 
fLoc.  cit. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        47 

cent,  aqueous  solution  of  water  soluble  yellow  esoin.  To  this 
mixture  add  70  c.c.  or  80  c.c.  of  a  i  per  cent,  aqueous  solu- 
tion of  methylene-blue  (Ehrlich  rectified).  Filter.  Dry  the 
residue  left  on  the  filter  paper,  powder  in  a  mortar.  About 
.7  to  i  gram  of  powder  is  thus  obtained.  Dissolve  in  about 
400  c.c.  of  pure  methyl  alcohol  (Merk). 

Nocht*  recommends  the  addition  of  silver  oxide  to  i  per 
cent,  methylene-blue  solution.  The  silver  oxide  in  this  case 
is  obtained  by  precipitating  i  gram  of  silver  nitrate  with  suf- 
ficient alkali,  and  adding  this  to  100  c.c.  of  the  i  per  cent, 
methylene-blue  solution,  and  allowing  the  mixture  to  ripen  for 
four  or  five  days  at  room  temperature. 

Goldhornf  recommends  the  use  of  lithium  carbonate  in- 
stead of  the  sodium  salt.  His  directions  are  to  boil  2  grams  of 
lithium  carbonate,  and  2  grams  of  methylene-blue  in  200  c.c.  of 
distilled  water.  This  is  done  in  a  double  boiler,  and  from  time 
to  time  samples  are  examined  in  test-tubes  to  see  whether  the 
polychrome  is  formed  as  indicated  by  a  red  color.  The  mix- 
ture after  cooling  spontaneously  is  filtered,  and  the  half  of  it 
is  rendered  distinctly  acid  with  acetic  acid.  The  two  halves 
are  then  poured  together.  The  rest  of  the  procedure  does  not 
differ  essentially  from  others  described  except  that  he  recom- 
mends commercial  wood  alcohol  in  the  place  of  pure  methyl 
alcohol  for  dissolving  the  stain. 

Wright's  modification  is  as  follows:  To  100  c.c.  of  a  i  per  cent,  solution  of 
sodium  bicarbonate  in  water  add  i  gram  of  methylene-blue.  Place  in  the 
steam  sterilizer  at  100°  C.  for  one  hour.  When  cool  add  one-tenth  per  cent, 
watery  solution  of  eosin  (Griibler,  yellowish,  soluble  in  water)  until  the  mixture 
loses  its  blue  color,  becomes  purple,  and  a  metallic  scum  forms  on  the  surface. 
About  500  c.c.  of  the  eosin  solution  are  needed.  Collect  the  precipitate  on  a 
filter;  let  it  dry;  make  a  saturated  solution  of  the  precipitate  in  methyl-alcohol; 
filter.  To  the  quantity  obtained  add  one-fourth  as  much  methyl  alcohol,  so 

*Enzyklopaedie  d.  Mikros.  Technik.,  quoted  by  Hastings,  Jour.  Exper. 
Med.,Vol.  7,  1905,  p.  266.  See  also  Duval.  Jour.  Exper.  Med.,  IX.,  1907, 
P-  381- 

\Jour.  Exper.  Med.,  Vol.  VIII.,  1907,  pp.  451-459. 


48  MANUAL  OF  BACTERIOLOGY 

that  the  solution  may  not  be  completely  saturated.  The  purpose  of  the  above 
procedures  is  to  modify  the  methylene-blue  so  that  other  staining  elements  are 
developed  in  it  (poly chromism) .  The  modified  methylene-blue  solution  is 
then  combined  with  eosin.  For  further  details  see  Wright.  Journal  of  Medical 
Research.  Vol.  VII.,  1902. 

Beside  the  various  modifications  of  the  Romano wsky  stain  given  above,  a 
number  have  been  recommended,  all  of  them  having  this  in  common  that  the 
nuclear  stain  depends  upon  the  modification  of  methylene-blue  due  to  the 
action  of  dilute  alkali.  Giemsa  has  put  on  the  market  a  ready  prepared  stain 
which  is  essentially  a  modified  Romanowsky.  The  trade  name  for  the  prep- 
aration is  "Asur  I.  (pur.)".  "Asur  II"  is  a  mixture  of  Asur  I  with  equal 
parts  of  methylene-blue. 

Staining  of  Spores. — The  method  is  applicable  to  cover- 
glass  preparations  which  may  be  prepared  in  the  usual  way 
from  material  containing  spores. 

(a)  After  drying  the  smear  on  the  cover-glass,  and  fixation 
with  heat  by  passing  through  the  flame  three  times,  use  aniline- 
water  fuchsin  or  carbol-fuchsin  as  a  stain. 

(b)  Heat  until  the  preparation  begins  to  boil;  remove  for  a 
minute;  heat  again,  and  again  remove;  repeat  this  process  six 
times. 

(c)  Wash  in  dilute  alcohol  (Novy)  or  in  a  weak  solution  of 
"acetic  or  hydrochloric  acid  for  a  few  seconds  to  a  minute. 
Some  spores  are  quickly  decolorized  by  i  per  cent,  acetic  acid; 
others  may  keep  the  stain  when  subjected  to  3   per  cent, 
hydrochloric  acid  alcohol  for  a  minute. 

(d)  Wash  in  water. 

(e)  Stain  with  watery  solution  of  methylene-blue  half  a 
minute. 

(/)  Wash. 

fe)   Dry. 

(h)  Balsam. 

The  spores  are  intensely  stained  by  the  fuchsin.  The  stain 
is  removed  from  everything  except  the  spores  by  the  acid. 
The  methylene-blue  solution  stains  the  bodies  of  the  bacteria, 
the  spores  remaining  brilliant  red.  There  are  various  other 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        49 

methods  for  staining  spores,  but  this  procedure  gives  good 
results.  The  principle  is  the  same  as  in  staining  the  tubercle 
bacillus,  except  that  more  pains  are  needed  to  impregnate 
spores  with  the  dye. 

The  spores  are  less  readily  stained  than  tubercle  bacilli,  and 
for  this  reason  require  prolonged  and  repeated  treatment  with 
the  hot  dye.  The  spores  also  give  up  the  stain  much  more 
readily  than  tubercle  bacilli,  and  consequently  will  not  retain 
the  stain  if  actively  decolorized.  Bacteria  sometimes  show 
granules  of  protoplasm  stained  by  the  methods  used  for  spores, 
but  these  granules  are  not  apt  to  be  mistaken  for  spores. 

Staining  of  Capsules. — The  capsules  which  many  bacteria 
possess  appear  to  be  made  of  gelatinous  substance,  which  is 
difficult  to  stain. 

Method  of  Welch. — (a)  Cover-glass  preparations  are  made 
in  the  usual  manner.  Pour  glacial  acetic  acid  over  the  film. 

(b)  After  a  few  seconds,  replace  with  aniline-water  gentian- 
violet,  without  washing  in  water.     Change  the  stain  several 
times  to  remove  all  the  acetic  acid.     Allow  it  to  act  three  or 
four  minutes. 

(c)  Wash  and  examine  in  salt  solution,  0.8  to  2.0  per  cent. 
Bacteria  are  deeply  stained,  while  their  capsules  are  pale 

violet.  This  method  has  been  recommended  for  staining  the 
capsule  of  the  pneumococcus. 

Methods  oj  Hiss* — i.  (a)  Make  cover-glass  preparations  in 
the  usual  manner,  and  fix  in  the  flame. 

(b)  Stain  for  a  few  seconds  in  a  half-saturated  watery  solution 
of  gentian-violet. 

(c)  Wash  in  0.25  per  cent,  solution  of  potassium  carbonate  in 
water. 

(d)  Mount  and  study  in  the  same. 

2.  (a)  Cover-glass  preparations  are  made  and  fixed  in  the 
ordinary  way. 

^Journal  of  Experimental  Medicine,  VI.,  1905,  p.  317. 
4 


50  MANUAL  OF  BACTERIOLOGY. 

(6)  Use  the  following  stain,  heated  till  it  steams: 

Saturated  alcoholic  solution  of  gentian-violet  or  fuchsin .    .        5  c.c. 
Distilled    water 95  c.c. 

(c)  Wash  in  20  per  cent,  solution  of  cupric  sulphate. 

(d)  Dry  and  mount  in  Canada  balsam. 

The  methods  of  Hiss  are  recommended  to  be  used  for  bac- 
teria that  have  been  cultivated  on  media  containing  blood- 
serum.  They  have  shown  that  many  streptococci  have 
capsules.  The  writer  has  had  good  success  from  the  latter 
method,  with  preparations  of  the  pneumococcus  from  animal 
tissues. 

Staining  of  Flagella.— Flagella  are  among  the  most  diffi- 
cult of  all  objects  to  stain.  The  best-known  method  is  that  of 
Loffler.  It  is  important  to  use  young  cultures,  preferably  the 
cloudy  water  in  an  agar  culture,  or  a  fresh  beef-broth  culture. 

(a)  Spread  a  small  portion  of  the  culture  on  a  cover-glass  using 
a  drop  of  water  if  necessary.     The  preparations  must  be  ex- 
ceedingly thin.     The  spreading  must  be  done  with  care  in  order 
not  to  break  off  the  delicate  flagella.     It  is  better  to  allow  the 
drop  of  culture  to  run  of  its  own  accord  over  the  cover-glass, 
and  not  stir  it  with  the  platinum  needle  more  than  is  absolutely 
necessary.     The    cover-glass    must    be    perfectly    clean    (see 
page  25). 

(b)  After  drying,  fixation  is  effected  by  passing  through  the 
flame  three  times. 

(c)  The  essential  point  in  this  method  is  the  use  of  a  mordant 
as  follows : 

Tannic  acid,  20  per  cent,  solution 10  c.c. 

Saturated  solution  of  ferrous  sulphate 5  c.c. 

Saturated  alcoholic  solution  of  fuchsin i  c.c. 

'» 

This  solution  is  filtered  and  a  few  drops  are  placed  on  the 
cover-glass,  or  the  cover-glass  is  placed,  face  down,  in  a  dish 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.        51 

containing  the  stain;  it  is  then  left  for  one  to  five  minutes, 
warming  slightly. 

(d)  Wash  in  water. 

(e)  Stain    with    aniline-water    fuchsin,    or    carbol-fuchsin. 
(/)  Wash  in  water. 

fe)   Dry. 

(h)  Mount  in  Canada  balsam. 

(According  to  Loffler,  certain  bacteria  require  the  addition 
of  an  acid  solution,  and  certain  others  an  alkaline  solution,  but 
many  observers  consider  this  unnecessary.) 

Another  and  very  valuable  method  is  that  of  Van  Ermengem. 

(a)  Make  and  fix  cover-glass  preparations  as  in  the  pre- 
ceding method. 

(b)  Use  the  following  mordant  for  one-half  hour  at  room- 
temperature  or  for  five  minutes  at  50°  to' 60°  C. 

Osmic  acid,  2  per  cent,  solution . i  part. 

Tannic  acid,  10  to  25  per  cent,  solution 2  parts. 

(c)  Wash  carefully  in  distilled  water  and  then  in  alcohol. 

(d)  Place  for  a  few  seconds  in  a  0.25  to  0.50  per  cent,  solution 
of  nitrate  of  silver — "the  sensitizing  bath." 

(e)  Without  washing  transfer  to  the  "reducing  and  rein- 
forcing bath:" 

Gallic  acid 5  grams. 

Tannic  acid 3  grams. 

Fused  potassium  acetate 10  grams. 

Distilled  water 350  c.c. 

(/)  Ater  a  few  seconds,  replace  the  preparation  in  the  nitrate 
of  silver  solution,  in  which  it  is  kept  constantly  moving,  till  the 
solution  begins  to  acquire  a  brown  or  black  color. 

Some  recommend  leaving  the  preparation  in  the  nitrate  of 
silver  solution  for  two  minutes  in  the  first  place,  and  in  the 
reducing  bath  for  two  minutes,  without  using  th*e  nitrate  of 
silver  solution  a  second  time. 


52  MANUAL  OF  BACTERIOLOGY. 

(g)  Finally  wash  in  distilled  water,  dry,  mount  in  Canada 
balsam.  It  is  difficult  to  avoid  the  formation  of  precipitates; 
otherwise  the  results  of  this  method  are  usually  good. 

A  number  of  other  methods  or  modifications  of  existing 
methods  have  been  recommended,  all  giving  more  or  less  satis- 
factory results.  The  flagella  are  rarely  or  never  stained  uni- 
formly throughout  the  preparation  by  any  method.  Those 
lying  at  the  edges  of  the  drop  are  most  apt  to  be  intensely 
stained. 


CHAPTER  III. 
STERILIZATION. 

BY  sterilization  is  meant  the  killing  of  all  microorganisms 
found  on  or  in  any  body  or  substance.  It  is  possible  to  sterilize 
objects  by  the  use  of  bichloride  of  mercury  (corrosive  subli- 
mate), carbolic  acid  and  other  chemical  agents,  but  their  value 
in  practice  is  often  overrated.  The  most  effective  sterilization 
is  that  done  with  heat,  either  by  direct  application  of  the  Bun- 
sen  burner,  or  by  heated  air,  or  by  steam,  or  by  boiling. 

The  naked  flame  of  the  Bunsen  burner  or  the  alcohol  lamp 
is  used  largely  for  the  sterilization  of  small  articles.  It  is 
evident  that  no  more  efficient  way  of  sterilization  could  be  de- 
vised than  by  burning  objects  or  subjecting  them  to  a  red  heat. 
The  uses  of  this  method  will  at  once  suggest  themselves;  for 
instance,  surgical  dressings  that  have  become  soiled  with  dis- 
charges and  similar  materials  can  be  most  easily  disposed  of  by 
simply  burning  them  up.  In  laboratory  work  the  flame  is  con- 
stantly employed  for  the  sterilization  of  the  platinum  wire, 
forceps,  pipettes  and  cover-glasses;  occasionally  test-tubes  are 
sterilized  in  this  manner. 

Hot-air  Sterilization. — Hot  air,  at  a  temperature  of  150° 
C.,  or  higher,  maintained  for  an  hour,  is  very  valuable  for  some 
materials,  although  less  effective  than  steam.  It  has  been 
found  that  the  spores  of  certain  bacteria  are  not  killed  even  by 
exposure  to  this  temperature,  but  it  is  sufficient  for  ordinary 
conditions.  Hot-air  sterilization  is  employed  for  glassware, 
such  as  Petri  dishes,  flasks  and  test-tubes.  Flasks  and  test- 
tubes  are  generally  plugged  with  raw  cotton.  The  heating 
should  not  be  allowed  to  go  to  the  extent  of  scorching  the 

•    53 


54 


MANUAL    OF    BACTERIOLOGY. 


cotton;  but  a  faint  light  browning  of  the  outside  is  permissible, 
and  is  an  indication  that  the  sterilization  is  effectual.  Glass- 
ware should  be  placed  within  the  sterilizer  when  it  is  cold,  and 
after  heating  should  be  allowed  to  cool  gradually  in  order  to 
avoid  breaking.  Hot-air  sterilization  is  never  used  for  culture- 
media. 

The  apparatus  used  for  hot-air  sterilization  consists  of  a  box 


FIG.  ii. — Hot-air  sterilizer. 


made  of  sheet-iron,  the  walls  being  double,  with  an  air-space 
between  them.  On  one  side  is  a  door.  There  are  openings  at 
the  top  to  secure  the  circulation  of  air  in  the  air-chamber.  A 
thermometer  passes  from  the  top  into  the  interior  of  the  sterili- 
zer so  that  one  may  read  off  the  temperature  that  is  being  at- 
tained. The  sterilizer  should  be  placed  so  that  there  will  be 
no  danger  of  its  setting  fire  to  inflammable  articles,  as  the  heat 


STERILIZATION.  55 

may  occasionally  become  very  intense.  It  is  well,  if  possible, 
to  have  it  fastened  to  a  brick  wall. 

Boiling. — Boiling  in  water  is  an  efficient  means  of  steriliza- 
tion for  some  purposes. 

Steam  Sterilization. — In  laboratory  work  "live"  steam 
or  steam  under  pressure  is  generally  substituted  for  simple 
boiling  in  the  water  and  is  more  effective  than  the  latter. 

By  "live"  steam  is  meant  the  steam  which  streams  off  from 
the  Koch  or  from  the  Arnold  sterilizer  described  below.  By 
steam  under  pressure  is  meant  that  generated  in  an  autoclave 
also  described  below.  But  in  both  cases  the  steam  to  be  ef- 
fective must  be  saturated  with  moisture.  In  the  Koch  and  in 
the  Arnold  sterilizers  this  is  always  the  case;  though  experi- 
ments have  been  made  in  which  the  steam  was  kept  at  100°  C., 
but  deprived  of  moisture,  and  in  this  case  it  was  much  less  ef- 
fective. It  becomes  under  such  circumstances  merely  air  with 
more  or  less  moisture,  and  experiments  have  shown  that  the 
sterilizing  power  of  heated  air  is  in  proportion  to  the  amount 
of  moisture  contained.  Steam  under  pressure  is  not  neces- 
sarily saturated  with  moisture.  If  water  is  boiled  in  a  closed 
vessel  the  steam  will  be  mixed  with  the  air  which  is  inclosed 
unless  this  is  allowed  to  escape  before  the  vessel  is  closed.  This 
matter  is  of  practical  importance  in  using  the  autoclave. 

Steam  is  employed  for  perishable  bodies  which  would  be 
injured  by  dry- air  sterilization  or  by  chemical  germicides;  for 
example,  it  is  used  for  surgical  instruments  and  for  culture- 
media;  in  laboratory  work,  especially  for  culture-media.  It 
has  been  found  that  there  are  some  forms  of  bacteria  which, 
in  the  resting  or  spore  stage,  can  resist  the  action  of  steam 
even  for  several  hours.  Such  prolonged  exposure  to  steam 
would  be  injurious  to  culture-media,  which  are  more  or  less  un- 
stable organic  substances.  What  is  called  fractional,  inter- 
mittent or  discontinuous  sterilization  is  used  for  such  materials. 
By  that  plan  the  medium  is  sterilized  with  steam  for  fifteen 


56  MANUAL    OF    BACTERIOLOGY. 

minutes  on  each  of  three  consecutive  days.  The  object  of 
intermittent  sterilization,  as  explained  by  Tyndall,  who  pro- 
posed it,  is  this:  The  culture-medium  may  be  supposed  to  con- 
tain fully  developed  bacteria,  and  also  bacteria  in  the  spore  or 
resting  stage.  The  first  sterilization  of  fifteen  minutes  will 
probably  be  sufficient  to  destroy  all  the  fully  developed  bacteria; 
during  the  twenty-four  hours  between  the  first  and  second 
sterilization  all  of  the  spores  which  have  survived  the  first 
sterilization  may  be  expected  to  have  become  fully  developed 
into  bacteria  which  can  be  destroyed  by  the  second  steriliza- 
tion; the  third  sterilization  is  directed  against  any  spore  forms 
which  may  possibly  have  survived  the  second  sterilization. 

Although  the  spore  forms  which  are  so  extremely  resistant 
are  non-pathogenic,  as  for  example  spores  of  the  hay  bacillus 
and  of  the  potato  bacillus,  they  nevertheless  are  capable  of 
ruining  the  culture-media  with  which  one  works. 

It  has  been  shown  by  Theobald  Smith  that  the  discontinuous 
method  cannot  be  relied  upon  to  sterilize  fluids  in  shallow  layers 
that  are  freely  exposed  to  the  air.  For  if  the  spores  of  anaero- 
bic bacteria  happen  to  be  present  in  such  fluids,  they  will  not 
develop  into  the  adult  form  between  the  applications  of  heat, 
under  aerobic  conditions. 

The  sterilization  of  culture-media  is  usually  effected  by 
seven  to  ten  minutes'  sterilization  in  the  autoclave  at  fifteen 
pounds'  pressure.  This  is  more  certain  than  the  fractional 
method,  and  may  be  employed  without  injury  for  all  media 
except  nutrient  gelatin.  The  gelatinizing  property  of  this  is 
interfered  with  by  the  high  temperature  of  the  autoclave.  It  is 
not  advised  to  sterilize  media  containing  sugars  in. the  auto- 
clave. 

The  form  of  sterilizer  widely  used  in  the  United  States  is  that 
which  is  known  as  the  Arnold  Steam  Sterilizer. 

The  Arnold  sterilizer  consists  of  a  cylinder  of  tin  or  copper 
with  a  cover,  which  is  enclosed  in  a  movable,  cylindrical  outer 


STERILIZATION. 


57 


cover  or  hood.  The  inner  cylinder  has  an  opening  in  the  bot- 
tom through  which  steam  may  enter,  the  steam  coming  from 
a  small  chamber  underneath  with  a  copper  bottom  to  which 
the  flame  is  applied.  The  peculiarity  of  this  form  of  sterilizer 
consists  in  the  fact  that  the  steam  which  escapes  from  the  ster- 
ilizing chamber  condenses  beneath  the  outer  cover  or  hood  and 
falls  back  upon  the  pan  over  the  chamber  in  which  the  steam  is 


FIG.  12. — Diagram  of  the  Arnold  steam  sterilizer. 

generated.  The  bottom  of  this  pan  is  perforated  with  three 
small  holes,  which  allow  the  water  of  condensation  to  return 
into  the  chamber  where  the  steam  is  generated.  The  sterilizer, 
therefore,  to  a  certain  extent,  supplies  itself  with  water,  al- 
though not  by  any  means  perfectly.  It  is,  however,  less  likely 
to  boil  dry  than  other  forms  of  sterilizers,  and  it  has  the  ad- 
vantage of  being  reasonably  cheap  and  quite  effective.  The 


58  MANUAL    OF    BACTERIOLOGY. 

space  inclosed  by  the  hood  also  serves  as  a  steam-jacket  and 
helps  to  overcome  fluctuations  in  temperature.  A  great  im- 
provement upon  the  ordinary  Arnold  sterilizer  is  the  modifica- 
tion of  it  devised  by  the  Massachusetts  Board  of  Health. 

In  the  use  of  this,  or  any  form  of  steam  sterilizer,  the  time  is 
noted  from  the  period  when  boiling  is  brisk  and  it  is  evident 
that  the  sterilizing  chamber  is  filled  with  hot  steam;  or,  what 


_ 

FIG.  13. — Steam  sterilizer,  Massachusetts  Board  of  Health. 

is  better,  when  the  thermometer  registers  100°  C.,  if  the  ster- 
ilizer be  provided  with  a  thermometer.  With  a  large  Arnold 
sterilizer  a  temperature  of  100°  C.  may  not  be  reached  until  it 
has  been  heated  with  a  rose-burner  for  twenty  to  thirty-five 
minutes.  When  bulky  articles  or  large  amounts  of  material 
are  to  be  sterilized  allowance  must  be  made  for  the  time  neces- 
sary to  bring  the  temperature  in  the  middle  of  the  mass  to 
100°  C. 


STERILIZATION. 


59 


The  sterilizer  invented  by  Koch  is  still  largely  in  use.  It  is 
a  tall,  cylindical,  tin  vessel  covered  with  asbestos  or  felt.  The 
lower  portion  is  filled  with  water;  on  the  side  is  a  water-gauge 
indicating  the  height  of  the  water,  in  order  that  one  may  ob- 
serve when  there  is  danger  of  the  sterilizer  boiling  dry.  Over 
the  top  there  is  a  tight-fitting  cover.  The  steam  is  generated 


FIG.  14. — Koch's  steam  sterilizer. 

by  a  Bunsen  burner  standing  underneath.  A  perforated  shelf 
placed  some  distance  above  the  surface  of  the  water  is  for  the 
reception  of  the  tubes  and  flasks  that  are  to  be  sterilized. 

The  sterilization  of  blood-serum  as  a  culture  medium  sometimes  has  to  be 
performed  in  a  specially  devised  sterilizer,  when  a  clear,  fluid  medium  is  desired. 
In  this  case  the  serum  is  heated  for  an  hour  on  each  of  six  consecutive  days  to 
a  temperature  of  only  58°  C.  To  obtain  a  transparent  but  solid  medium  the 
serum  is  kept  at  a  temperature  of  75°  C.  for  an  hour  on  each  of  four  consecutive 
days.  The  process  must  be  conducted  carefully  to  avoid  clouding  of  the 


60  MANUAL    OF    BACTERIOLOGY. 

Pasteurization. — The  name  pasteurization  has  been  ap- 
plied to  the  partial  sterilization  of  substances  at  a  compara- 
tively low  temperature.  It  is  employed  particularly  for  milk. 
Although  the  temperature  used  (63°  to  65°  C.  for  20  to  30 
minutes)  is  sufficient  to  destroy  all  ordinary  pathogenic 
bacteria,  at  least  in  test-tube  experiments,  and  the  probability 
is  that  where  the  bacteria  are  actually  brought  to  the  tempera- 
tures indicated  they  are  destroyed  in  milk,  too  great  reliance 
should  not  be  placed  upon  this  mode  of  sterilization;  for,  as 
elsewhere  stated,  under  some  circumstances  milk  may  afford 
a  protection  for  the  bacteria,  and  this  should  be  borne  in 
mind  particularly  in  regard  to  tuberculosis  and  typhoid  fever. 
Furthermore,  the  great  majority  of  the  saprophytic  bacteria  are 
destroyed,  and  milk  which  has  been  pasteurized  will  remain  un- 
changed for  several  days,  if  kept  cool.  Its  application  is 
principally  in  the  feeding  of  infants  in  cases  where  raw  milk 
causes  digestive  disturbances.  Freeman*  has  invented  a  pail 
of  special  form  for  the  pasteurization  of  milk  in  bottles.  This 
pail  is  filled  with  hot  water  and  the  bottles  are  placed  in  it;  it  has 
been  found  to  keep  up  a  temperature  of  about  75°  C. 

The  Autoclave. — The  autoclave  is  an  apparatus  designed 
for  sterilization  by  steam  under  pressure.  It  was  invented  in 
France,  but  is  now  used  extensively  in  all  parts  of  the  world. 
Steam  generated  at  the  ordinary  atmospheric  pressure  is  much 
less  destructive  to  bacteria,  and  especially  to  their  spores,  than 
steam  in  the  autoclave  at  a  pressure  of  an  additional  one-half 
to  one  atmosphere,  yj  to  15  pounds;  the  steam  then  reaches 
a  temperature  of  about  112°  to  120°  C.  Under  these  condi- 
tions culture-media  may  be  sufficiently  sterilized  in  the  auto- 
clave in  ten  minutes,  and  at  a  single  sterilization.  The  auto- 
clave consists  of  a  metal  cylinder  with  a  movable  top,  which 
is  fastened  down  tightly  during  sterilization.  It  is  furnished 

*Medical  Record.  July  2,  1892,  and  August  4,  1894.  This  pail  is  sold  by 
James  T.  Dougherty,  411  West  Fifty-ninth  Street,  New  York. 


STERILIZATION. 


6l 


with  a  thermometer,  a  pressure  gauge,  a  stop-cock,  and  a 
safety-valve  which  is  set  to  allow  the  steam  to  escape  when 
the  desired  pressure  is  attained  and  thus  prevents  it  from 
running  too  high.  Heat  is  furnished  by  a  gas-burner  under- 
neath. The  lower  part  of  the  cylinder  contains  water.  The 
objects  to  be  sterilized  are  supported  above  this  water  on  a 
perforated  bottom  or  shelf. 


FIG.  15. — Autoclave. 


[t  is  necessary  to  follow  certain  precautions  in  the  use  of 
the  autoclave.  Allusion  has  already  been  made  to  the  neces- 
sity for  having  the  steam  saturated  with  moisture.  This  is  ef- 
fected by  allowing  the  air  to  escape  after  the  heat  is  applied, 
and  in  order  to  be  sure  that  all  the  air  has  really  been  expelled, 
the  stop -cock,  with  which  all  autoclaves  are  provided,  is 
left  open  until  the  steam  escapes  freely.  The  stop-cock  is  then 


62 


MANUAL    OF    BACTERIOLOGY. 


closed,   and  the  pressure  begins  to  rise.     After  leaving  the 
articles  to  be  sterilized  in  the  autoclave  for  the  length  of  time 
desired,  the  apparatus  must  not  be  opened  while  the  steam 
contained  within  it  is  still  under  pressure,  as  there  may  be  a 
sudden  evolution  of  steam  upon  the  removal  of  the  pressure 
which  may  blow  the  media  out  of  their  tubes  and  flasks.     On 
the  other  hand,  the  pressure  must  not  be  allowed  to  drop  below 
zero,  for  in  this  case  the  plugs  of  the 
tubes  or  flasks  may  be  sucked  in.     The 
apparatus  must,  therefore,  be  kept  closed 
until  the  gauge  shows  that  the  atmos- 
pheric pressure  is  as  great  as  the  pressure 
within,  or,  what  is  equivalent,  until  the 
temperature  has  fallen  to   100°  C.  but 
not   below   this   temperature.      A    good 
rule   is   to   watch   until  the  pressure  is 
very    near    zero,    and    the    temperature 
very  near  100°  C.,  and  then  to  cautiously 
open  the  stop-cock  a  very  little.     The 
autoclave  may  be  opened  as  soon  as  the 
hissing  from  the  out-driven  steam  ceases. 
Gelatin  may  be  damaged  by  sterilization 
with  the  autoclave,  if  it  be  heated  too 
long  or  too  high  a  temperature.      Media 
containing  sugar  should  not  be  sterilized 
in  the  autoclave  (see  page  67). 

Sterilization  by  Filtration. — Ordinary  niters  are  useless 
for  this  purpose,  but  the  tubes  or  bougies  of  unglazed  porcelain  I 
devised  by  Pasteur  and  Chamberland  are  effective  when  prop-  | 
erly  employed.     They  are  made  in  several  different  grades  of 
porosity.     In  the  Berkefeld  filter  bougies  made  of  infusorial 
earth  are  used,  and  the  pores  in  this  are  larger  than  those  of  the 
Pasteur  filter.   The  coarser  of  these  filters  permits  the  passage  ! 
of  very  small  bacteria.     Bacteria  of  average  size,  like  bacillus  , 


FIG.  1 6. — Kitasato 
filter. 


STERILIZATION.  63 

coli  communis,  may  grow  through  the  pores  in  the  walls  of  both 
the  Berkefeld  and  Pasteur  filters  if  sufficient  nutrient  material 
is  present  to  permit  of  their  multiplication.* 

Filters  of  these  kinds  are  widely  used  for  water,  and  will  be 
spoken  of  in  connection  with  the  chapter  on  water.  Similar 
tubes  are  employed  for  the  nitration  of  certain  organic  nutrient 
media  whose  ingredients  would  be  damaged  by  sterilization 
with  heat,  chiefly  extracts  of  organs.  The  soluble  * 'toxins" 
of  bacteria  may  be  obtained  by  nitration  of  fluid-cultures 
through  such  tubes,  which  remove  the  bacteria  (Fig.  16) 
These  fluids  usually  filter  very  slowly,  and  filtration  has  to  bo 
assisted  by  some  form  of  vacuum-pump;  usually  the  filter- 
pump,  which  is  used  in  connection  with  a  stream  of  running 
water,  is  employed.  Compressed  air  or  carbonic  acid  may  be 
used  to  assist  in  forcing  fluids  through  the  filter.  The  filter 
bougies,  the  flasks  and  all  parts  of  the  apparatus  must,  of 
course,  be  sterilized  by  heat  before  and  after  using. 

*Wherry.     Journal  of  Medical  Research.     Vol.  VIII.,  1902. 


CHAPTER    IV. 
CULTURE-MEDIA. 

CULTURE-MEDIA  are  substances  in  which  bacteria  are  art  in 
daily  cultivated.  The  number  of  such  substances  is  very 
large,  different  materials  being  suited  to  different  purposes  and 
to  different  kinds  of  bacteria.  The  most  important  ones  are 
nutrient  bouillon  or  beef-tea,  nutrient  gelatin,  and  nutrient 
agar-agar.  The  two  last  have  a  jelly-like  consistency,  owing 
to  the  addition  of  a  gelatinizing  substance,  but  otherwise  are  of 
the  same  composition  as  the  bouillon. 

In  all  cases  the  media  must  be  either  free  from  bacteria 
originally  or  they  must  be  rendered  free  from  bacteria  in  order 
that  the  organisms  under  cultivation  may  be  studied  in  pure 
cultures.  This  is  effected,  as  a  rule,  by  steaming  in  the  steam 
sterilizer  or  in  the  autoclave.  For  special  purposes  nitration 
through  porcelain  filters  is  resorted  to. 

Preparation  of  Media. 

NUTRIENT    BOUILLON. 

Beef-extract  (such  as  Liebig's) 3  grams. 

Peptone,  pure  (Witte's)* 10  grams. 

Sodium  chloride  (common  salt) 5  grams. 

Water i  liter. 

The  solid  ingredients  are  dissolved  in  water,  and  the  mixture 
is  boiled  for  a  few  minutes.  It  is  made  neutral  or  very  faintly 
alkaline  by  the  addition  of  a  solution  of  sodium  hydroxide, 
drop  by  drop,  the  reaction  being  tested  at  intervals  with 
litmus-paper.  As  soon  as  the  proper  reaction  is  reached,  it  is 

*Commercial  "peptones"  are  mixtures  of  albumose  and  a  small  amount  of 
peptone. 

64 


CULTURE-MEDIA.  65 

filtered  through  filter-paper.  The  filter-paper  should  be 
folded  and  creased  as  is  done  by  pharmacists;  it  is  in  the 
usual  manner  placed  in  a  glass  funnel,  and  should  be  moistened 
with  water  before  using.  After  filtration  the  medium  is  to  be 
placed  in  properly  plugged  tubes  or  flasks,  and  is  to  be  sterilized 
once  in  the  autoclave,  or  in  the  steam  sterilizer  for  fifteen  min- 
utes or  longer  on  each  of  three  consecutive  days.  When  pre- 
cipitates form,  they  are  usually  caused  by  a  too  alkaline  reac- 
tion. That  may  be  corrected  by  the  addition  of  a  little  weak 
hydrochloric  acid,  drop  by  drop,  testing  frequently  with  litmus- 
paper. 

A  more  accurate  way  of  obtaining  the  proper  reaction  is  Schultz's  method. 
Take  of  the  bouillon  10  c.c.;  add  a  few  drops  of  phenolphthalein*  (alcoholic 
solution,  §  per  cent.);  with  a  burette  add,  drop  by  drop,  a  solution  of  caustic 
soda,  0.4  per  cent.,  until  a  faint  red  color  appears,  which  indicates  the  beginning 
of  the  alkaline  reaction.  This  procedure  is  followed  with  three  samples.  The 
amount  of  soda  solution  required  in  each  case  is  noted  and  the  average  taken. 
If  now,  on  the  average,  for  each  10  c.c.  of  bouillon  i  c.c.  of  soda  solution  needs 
to  be  added,  for  1000  c.c.  of  bouillon  100  c.c.  of  the  soda  solution  must  be  added ; 
only,  instead  of  adding  a  weak  soda  solution,  one-tenth  as  much  is  taken  of  a 
solution  ten  times  as  strong. 

Another  method  of  making  bouillon  and  that  most  usually 
recommended,  is  to  use,  instead  of  beef-extract,  500  grams 
(one  pound)  of  finely  chopped,  lean  beef,  which  is  placed  in  one 
liter  of  water  and  kept  on  ice  for  twenty-four  hours.  It  is 
strained,  thoroughly  cooked  to  coagulate  the  albumen  in  it, 
filtered  and  a  liter  of  fluid  obtained,  adding  water  if  necessary. 
The  peptone  and  salt  are  then  added  and  the  medium  heated 
to  dissolve  them.  Some  advise  the  addition  of  the  peptone  and 
salt  to  the  fresh  watery  extract  of  the  meat  before  boiling.  In 
preparing  media  for  the  purpose  of  water  analysis  it  is  advised 
to  leave  out  the  salt.  It  is  then  neutralized,  filtered  and  steril- 
ized. Although  bouillon  made  with  solid  beef-extract  is  con- 

*In  neutralizing  an  acid  culture-medium  it  has  been  found  that  when  the 
medium  appears  to  be  neutral  or  slightly  alkaline  to  litmus,  it  may  still  be 
acid  if  phenolphthalein  be  employed  as  an  indicator.  Fuller.  Journal  Ameri- 
can Public  Health  Association.  1895. 

5 


66  MANUAL    OF    BACTERIOLOGY. 

venient  and  serviceable  for  most  purposes,  it  is  advisable  to  use 
meat  when  the  bouillon  is  to  be  employed  for  the  development 
of  bacterial  toxins.  Meat  should  also  be  used  in  the  prepara- 
tion of  either  bouillon,  gelatin  or  agar-agar  when  new  species 
of  bacteria  are  being  studied  for  publication. 

In  both  of  these  cases  the  recommendations  of  the  American  Public  Health 
Association  should  be  followed.* 

These  recommendations  have  been  largely  followed  in  the  directions  for  the 
preparation  of  culture-media  given  below.  The  student  is  referred  to  the  re- 
port itself  for  further  details  than  those  given  below. 

The  following  solutions  are  required:  £  per  cent,  phenolphthalein  in  50 
per  cent,  alcohol,  normalf  (-^-)  and  twentieth  normal  (y^-)  solutions  of  sodium 
hydroxide  and  of  hydrochloric  acid. 

To  5  c.c.  of  bouillon  in  a  porcelain  evaporating  dish  add  45  c.c.  of  distilled 

-water;  boil  three  minutes;  add  i  c.c.  of  phenolphthalein  solution,  and  proceed 

with  the  titration  while  still  hot.     As  the  reaction  will  usually  be  found  acid, 

add  from  a  burette  —^  sodium  hydroxide   solution,  stirring  constantly,  until 

a  decided  pink  color  develops  in  the  entire  solution.     The  color  reaction  indi- 


*Report  of  Committee  of  the  Am.  Pub.  Health  Assn.  on  Standard  Methods 
of  Water  Analysis,  Journ.  Infect.  Dis.  Sup.  No.  i.  May,  1905. 

fA  normal  solution  of  any  substance  contains,  in  a  liter,  as  many  grams  of 
the  substance  as  there  are  units  in  its  molecular  weight,  in  case  it  contains  a 
single  atom  of  replaceable  hydrogen.  If  it  has  two  atoms  of  replaceable  hydro- 
gen the  number  of  grams  used  equals  the  molecular  weight  divided  by  two; 
and  so  on.  Thus  the  molecular  weight  of  sodium  hydroxide  is  40,  and  its  nor- 
mal solution  contains  40  grams  of  sodium  hydroxide  in  a  liter.  It  is  not 
expedient  to  prepare  normal  solutions  of  sodium  hydroxide  by  weight.  For 
convenience,  crystallized  oxalic  acid  is  used  as  a  starting  point  in  making  nor- 
mal solutions.  Its  molecular  weight,  including  a  molecule  of  water  of  crystal- 
lization, is  126.  As  it  is  a  dibasic  acid  (having  two  atoms  of  replaceable 
hydrogen),  half  of  this  weight,  or  63  grams,  per  liter,  is  taken.  Any  —--  acid 
solution  will  exactly  neutralize  an  equal  volume  of  any  — -  alkaline  solution. 
To  make  -~-  sodium  hydroxide  solution,  add  about  41  grams  of  pure  caustic 
soda  to  a  liter  of  distilled  water.  Find  the  amount  of  this  solution  needed  to 
exactly  neutralize  i  c.c.  of  -^-  solution  of  oxalic  acid;  this  amount  contains 
the  quantity  of  sodium  hydroxide  which  should  be  present  in  i  c.c.  of  a  nor- 
mal solution.  It  is  now  possible  to  calculate  the  amount  of  distilled  water  to 
be  added  in  order  that  i  c.c.  of  the  sodium  hydroxide  solution  may  neutralize 
i  c.c.  of  the  ~-  solution  of  oxalic  acid.  With  an  ~-  solution  of  sodium  hydrox- 
ide as  a  standard,  an  ~-  solution  of  hydrochloric  acid  may  be  prepared. 
Twentieth  normal  solutions  have  one-twentieth  the  strength  of  normal  solutions. 


CULTURE-MEDIA.  67 

cates  the  more  or  less  arbitrarily  adopted  neutral  point.  Repeat  this  procedure 
with  three  different  portions  of  bouillon,  and  determine  the  average  amount 
of  -^--jj-  sodium  hydroxide  required.  It  is  now  possible  to  calculate  the  amount 
of  -y-  sodium  hydroxide  needed  to  neutralize  the  whole  quantity  of  bouillon. 
This  should  be  added.  The  bouillon  should  then  be  boiled  for  ten  minutes, 
and  again  titrated.  It  will  usually  be  found  acid.  The  deficiency  should  be 
corrected  by  adding  the  necessary  amount  of  —--  sodium  hydroxide.  It  should 
be  boiled  again,  and  again  titrated,  and  any  deficiency  made  good.  It  is  rarely 
necessary  to  repeat  the  process,  except  to  determine  that  the  neutral  point  has 
been  reached.  After  neutralizing  it  is  boiled  thirty  minutes  and  filtered. 
Enough  -y-  hydrochloric  acid  or  sodium  hydroxide  is  added  to  give  the  degree 
of  acidity  or  alkalinity  desired.  It  is  then  sterilized. 

An  acid  reaction  may  be  denoted  by  +,  an  alkaline  by .  The  degree  of 

acidity  or  alkalinity  may  be  indicated  by  the  amount  of  — -  solution  required 
to  render  the  medium  neutral  to  phenolphthalein,  thus  +  i.oo  signifies  that  a 
medium  is  acid,  and  requires  i.oo  per  cent,  of  -y-  sodium  hydroxide  to 
neutralize  it. 

A  reaction  of  -f-  i.oo  is  recommended  as  the  optimum.  There  is  much  dis- 
agreement as  to  what  reaction  is  most  favorable  for  the  growth  of  the  majority 
of  species  of  bacteria.  Even  +  0.5  may  be  better  for  some  bacteria.  In  any 
case  the  degree  of  reaction  should  be  noted  in  descriptions. 

Bouillon  may  be  modified  by  the  addition  to  it  of  other  sub- 
stances, the  most  important  of  which  are  glycerin  (6  per  cent.) 
and  sugars, — as  dextrose,*  saccharose  or  lactose  (  i  per  cent.). 
It  is  better  to  sterilize  media  containing  sugars  in  the  steam 
sterilizer  by  the  fractional  method  rather  than  in  the  autoclave, 
for  the  reason  that  at  the  high  temperature  of  autoclave  decom- 
position of  the  sugars  may  occur. 

Sugar-free  Bouillon. — Ordinary  bouillon  often  contains  some  muscle-sugar, 
which  is  objectionable  if  fermentation  tests  with  lactose  or  saccharose  are 
to  be  made.  Muscle-sugar  must  also  be  removed  from  the  beef-juice  in 
.  growing  diphtheria  cultures  for  the  production  of  antitoxine.  To  secure  bouillon 
free  of  sugar,  the  water  is  added  to  the  finely  chopped  beef  as  in  other  cases,  but 
it  is  then  inoculated  at  once  before  any  further  preparation  with  a  culture  of 
B.  coli  communis  and  allowed  to  stand  in  the  incubator  for  twelve  or  fifteen  hours. 
Early  next  morning  it  is  boiled,  filtered,  peptone  and  salt  added,  and  the 
bouillon  is  prepared  as  usual. f  The  medium  should  be  tested  for  the  presr 

*Dextrose  is  the  principal  ingredient  of  commercial  grape-sugar  or  glucose 
and  should  be  obtained  in  a  pure  condition. 

fSee  Theobald  Smith.  Journal  o/ Experimental  Medicine.  Vol.  II.,  1901,  p.  546. 


68  MANUAL    OF    BACTERIOLOGY. 

ence  of  indol  before  it  is  used  for  diagnostic  purposes;  since  Rivas*  finds  that 
in  fermenting  meat-juice  with  B.  coli  by  Smith's  method  for  sugar-free  broth, 
indol  may  be  formed. 

Beef-extract 3  grams. 

Pepton 10  grams. 

Sodium  chloride 5  grams. 

Gelatin  (best  gold  label) 100  grams. 

Water i  liter. 

Dissolve  the  ingredients  in  the  water,  stirring  actively  to 
prevent  burning  at  the  bottom.  It  is  best  to  conduct  the  opera- 
tions in  granite  or  enamel-ware  vessels  over  a  large  Bunsen 
or  rose-burner.  Neutralize  with  sodium  hydroxide  solution 
(see  page  66).  Litmus-paper  or  titration  may  be  used  for 
testing.  The  reaction  at  the  beginning  will  usually  be  found 
to  be  quite  acid.  Allow  the  mixture  to  cool  until  below  60° 
C.,  and  add  the  whites  of  one  or  two  eggs  which  have  been 
beaten  up  with  a  little  water;  stir  in  thoroughly.  Heat  the 
mixture  to  the  boiling-point;  stir  at  the  bottom  to  prevent 
burning  and  at  the  same  time  avoid  as  far  as  possible  breaking 
the  coagulum  of  egg-albumen  which  forms  at  the  surface. 
Boil  for  ten  minutes.  Filter  while  hot.  The  nitration  may  be 
done  through  folded  filter-paper  which  has  been  moistened. 
It  is  well  to  fasten  a  piece  of  coarse  cheese-cloth  over  the  top  of 
the  funnel  to  catch  the  large  particles  of  coagulated  albumen. 
Place  in  suitable  tubes  or  flasks  plugged  with  cotton,  and 
sterilize  once  in  the  autoclave,  or,  preferably,  in  the  steam 
sterilizer  for  fifteen  minutes  on  each  of  three  consecutive  days. 
Gelatin  is  injured  by  too  prolonged  boiling  and  loses  its  solidi- 
fying qualities.  The  remarks  on  pages  66  to  67  with  regard  to 
the  use  of  beef  and  the  titration  method  for  the  preparation  of 
bouillon  apply  equally  to  gelatin. 

Instead  of  filter-paper,  some  prefer  to  filter  through  several 
layers  of  absorbent  cotton  placed  inside  of  the  moistened  glass 
funnel,  the  top  of  which  is  covered  with  coarse  cheese-cloth. 

^Journal  of  Infectious  Diseases.    Vol.  IV.,  No.  4,  Nov.  15,  1907,  pp.  641-646. 


CULTURE-MEDIA.  69 

This  expedient  answers  very  well,  but  nitration  through  paper 
is  apt  to  give  better  results. 

If  the  product  appears  cloudy  after  it  has  been  sterilized, 
it  may  be  that  the  egg-albumen  was  incompletely  coagulated 
in  the  first  place  or  that  the  reaction  has  been  made  too  alkaline. 
In  any  case  it  will  be  desirable  to  melt  it  and  filter  a  second 
time,  correcting  the  reaction  with  hydrochloric  acid  if  neces- 
sary. It  may  be  well  to  stir  in  another  egg  to  entangle  the 
opaque  particles;  then  to  boil  a  second  time  and  filter. 

The  medium  is  sometimes  modified  by  adding  to  it  other 
substances,  as  sugar,  glycerin,  etc.  The  solidifying  property 
of  the  gelatin  must  be  carefully  guarded,  and  too  much  boiling 
is  to  be  avoided.  Certain  bacteria,  it  will  be  found,  have  the 
property  of  causing  gelatin  to  become  permanently  liquid:  this 
is  called  liquefaction  or  peptonization.  Gelatin  melts  at  about 
25°  C.  and  solidifies  at  about  10°  C.  It  cannot  be  used  in  the 
incubator,  where  it  would  melt  at  the  temperature  of  38°  C. 
In  hot  weather  it  may  be  necessary  to  use  150  grams  of  dry 
gelatin  to  the  liter.  Nutrient  gelatin  is  usually  spoken  of 
simply  as  "gelatin." 

Nutrient  Agar-agar. — Agar-agar  (French,  gelose)  is  a  kind 
of  vegetable  gelatin  which  comes  from  the  southern  and  east- 
ern coast  of  Asia.  It  melts  with  much  greater  difficulty  than 
gelatin,  and  remains  solid  at  much  higher  temperatures.  In 
this  respect  it  behaves  very  peculiarly,  since  it  will  not  melt 
unless  it  is  heated  to  about  80°  C. ;  but  after  it  is  once  melted 
it  remains  fluid  at  40°  C.,  or  over.  After  it  solidifies  it  has 
to  be  heated  up  to  about  80°  C.  before  it  will  melt  again. 

The  medium  is  not  quite  transparent.  The  finished  me- 
dium is  commonly  called  "agar." 

Beef-extract 3  grams. 

Peptone      10  grams. 

Sodium  chloride 5  grams. 

Dry  Agar 15  grams. 

Water  .    .  i  liter. 


70  MANUAL    OF    BACTERIOLOGY. 

The  dry  agar,  cut  fine,  is  to  be  dissolved  in  water  over  a 
flame  or  in  the  autoclave.  It  should  be  boiled  for  from  one- 
half  hour  to  two  hours,  skimming  off  the  scum  which  forms 
on  the  surface  from  time  to  time.  The  beef-extract,  peptone 
and  sodium  chloride  are  dissolved  in  a  liter  of  water,  boiled  and 
neutralized.  Add  the  agar  now  in  solution  in  a  small  quantity 
of  water.  The  reaction  of  the  agar  alone  is  faintly  alkaline. 
Mix  thoroughly;  the  bulk  of  the  mixture  is  a  little  more  than  a 
liter,  and  should  be  reduced  to  a  liter  by  the  subsequent  boiling. 
Cool  to  about  60°  C.;  stir  in  the  whites  of  one  or  two  eggs  and 
boil  thoroughly.  Avoid  breaking  the  coagulum  of  egg  which 
is  designed  to  entangle  the  solid  particles  that  make  the  medium 
cloudy;  stir  at  the  bottom,  however,  to  prevent  burning. 
Filter  while  hot,  using  filter-paper  or  absorbent  cotton  covered 
with  cheese-cloth.  The  hot-water  funnel  originally  devised  for 
the  filtration  of  agar  is  not  necessary.  If  filtration  is  slow,  the 
funnel  and  flask  may  be  placed  inside  of  the  steam  sterilizer 
and  kept  heated  during  filtration.  The  medium  is  collected  in 
suitable  flasks  or  tubes  plugged  with  cotton,  and  sterilized  once 
in  the  autoclave  or  in  the  ordinary  steam  sterilizer  for  fifteen 
minutes  on  each  of  three  consecutive  days.  As  agar  is  fre- 
quently used  for  smear-cultures  where  a  slanted  medium  is 
desired,  some  of  the  tubes  may  be  allowed  to  cool  in  a  slantiug 
position.  It  is  not  well  to  keep  on  hand  many  tubes  which 
have  been  slanted,  as  the  medium  dries  more  rapidly.  Agar  is 
seldom  liquefied  by  bacteria,  though  a  few  bacteria  possess  the 
power  of  doing  this.  Its  solidifying  qualities  are  impaired 
somewhat  if  the  reaction  be  acid. 

The  remarks  on  pages  66  to  67  with  regard  to  the  use  of 
beef  and  the  titration  method  for  the  preparation  of  bouillon 
apply  equally  to  agar-agar. 

Glycerin-agar  is  used  extensively.  It  is  agar,  made  as 
above  directed,  to  which  6  per  cent,  of  glycerin  is  added  before 


. 

UNIVERSITY 

71 


sterilization.  It  is  very  useful  in  cultivating  the  bacilli  of 
tuberculosis  and  diphtheria. 

Sugar  -agar.  —  Before  sterilizing,  i  per  cent,  of  either  dextrose, 
lactose,  saccharose  or  other  sugars  may  be  added  to  agar. 
In  this  case  the  agar  should  be  prepared  from  sugar-free  broth 
as  in  the  preparation  of  broth  to  which  the  sugar,  are  added. 
With  media  containing  sugar,  litmus  forms  a  useful  indicator 
of  the  production  of  acid.  Enough  tincture  of  litmus  is  used 
to  give  the  medium  a  blue  color  before  sterilization;  the  litmus 
is  somewhat  unstable  and  prone  to  change  its  color  during 
sterilization.  Azolitmin  is  now  recommended  in  place  of 
litmus  (see  below).  Neutral  red  may  also  be  added  in  the 
same  manner;  its  color  is  changed  by  certain  bacteria  and 
not  by  others  (see  bacillus  of  typhoid  fever  and  bacillus  coli 
communis,  Part  IV.).  To  i  liter  of  nutrient  agar,  add  i  gram 
of  dextrose  and  0.05  gram  or  10  c.c.  of  a  saturated  aqueous 
solution  of  neutral  red.  Sterilize  as  usual. 

The  committee  on  Standard  Methods  of  Water  Analysis, 
Am.  Pub.  Health  Assn.*  recommend  the  following:  Lean 
meat  should  be  used,  not  beef  extract,  as  a  basis  for  the  various 
media;  sodium  chloride  shall  not  be  used  for  water  analysis; 
Witte's  peptone  shall  be  used;  gelatin  must  be  the  best  French 
brand,  and  the  10  per  cent,  solution  after  its  preparation  as  a 
culture  medium  shall  not  soften  at  25°  C.  All  sugars  shall  b,e 
chemically  pure,  glycerin  when  used  must  be  double  distilled. 
In  place  of  litmus,  where  this  is  used,  azolitmin  shall  be 
substituted. 

Potato.  —  The  potatoes  are  washed,  a  slice  is  removed 
from  each  end,  and  with  an  apple-corer  or  cork-borer  a  cylinder 
is  cut  out.f  This  cylinder  is  divided  diagonally  into  two  pieces. 

*Jour.  Inf.  Diseaes,  Supplement  No.  i,  May,  1905,  p.  104. 

fBolton.  The  Medical  News.  Vol.  L,  1887,  p.  318.  A  Method  of  Pre- 
paring Potatoes  for  Bacterial  Cultures.  Roux.  De  la  Culture  sur  Pomme  de 
Terre.  Annales  de  I'lnstitut  Pasteur.  T.  II.,  1888,  p.  28.  Globig.  Ueber 
einen  Kartoffel-Bacillus  mit  ungewohnlich  widersland;fahigen  Sporen.  Zeit- 
schrijt  jur  Hygiene,  p.  322,  1888. 


MANUAL    OF    BACTERIOLOGY. 


The  pieces  are  washed  in  running  water  for  several  hours. 
They  are  placed  in  test-tubes  containing  a  little  water  to  keep  the 
potato  moist,  and  are  supported  from  the  bottom  on  a  piece  of 
glass  tubing  about  i  to  2  cm.  in  length  (or  on  cotton,  or  in  a 
specially  devised  form  of  tube  with  a  constriction  at  the  bot- 
tom). The  tubes  are  plugged,  and  sterilized  as  with  other 
media.  Sterilization,  however,  must  be 
thorough  on  account  of  the  danger  of  con- 
tamination with  the  extremely  resistant  spores 
of  the  potato  bacillus.  Potato  is  best  when 
freshly  prepared;  it  is  likely  to  become  dry 
and  discolored  with  keeping.  It  is  a  very 
useful  ^medium;  certain  growths  on  it,  like 
those  of  the  bacillus  of  typhoid  fever  or  of 
glanders,  and  those  of  chromogenic  bacteria, 
are  very  characteristic. 

Milk. — Milk  fresh  as  possible  is  placed  in 
a  covered  jar,  sterilized  for  fifteen  minutes, 
and  then  kept  on  ice  for  twenty-four  hours. 
At  the  end  of  that  time  the  middle  portion  is 
removed  by  means  of  a  siphon.  The  upper 
and  lower  layers  must  not  be  taken;  the 
upper  part  contains  cream,  and  the  lower 
part  particles  of  dirt,  both  of  which  are  to  be 
avoided. 

The  reaction  should  be  corrected  to  -f  i 
if  the  milk  is  found  to  be  too  acid.  About  7 
to  10  c.c.  are  to  be  run  into  each  test-tube.  The  tube  is 
plugged  with  cotton  and  sterilized  as  usual.  When  milk  is 
contaminated  with  spores  of  the  hay  or  potato  bacillus  it  is 
sometimes  very  difficult  to  sterilize,  a  fact  of  much  importance 
in  connection  with  the  feeding  of  children,  where  the  frac- 
tional method  of  sterilization  and  the  use  of  the  autoclave  are 
impracticable. 


FIG.  17.— Tube 
containing  potato. 


CULTURE-MEDIA. 


73 


The  coagulation  of  milk,  which  is  brought  about  by  certain 
bacteria,  is  a  very  valuable  differential  point.  Litmus  milk  is 
prepared  as  above  and  has  added  to  it  i  per  cent,  of  azolitmin 
before  sterilizing.  This  indicates  whether  or  not  acids  are 
formed  by  the  bacteria  which  are  afterward  cultivated  in  the 
milk. 

DUNHAM'S  PEPTONE  SOLUTION. 

Peptone 10  grams. 

Sodium  chloride 5  grams. 

Water i  liter. 

Boil,  filter,  sterilize  in  the  usual  manner. 

Nitrate  Broth. — Dissolve  i  gram  of  peptone  in  1000  c.c. 
of  tap  water,  and  add  2  grams  of  nitrite-free  potassium  nitrate. 
This  solution  is  distributed  into  test-tube,  10  c.c.  in  each  tube. 

Broth  for  the  Indol  Test. — Standard  broth  described  above 
may  be  used  for  this  test  provided  it  contains  no  muscle-sugar, 
the  muscle-sugar  having  been  removed  by  cultivating  B.  coli  in 
the  beef  infusion  for  twenty-four  hours  previous  to  its  prepara- 
tion. Or  the  following  solution  may  be  employed  for  the  test: 

Dunham's  solution  is  valuable  to  test  the  development  of 
indol  by  bacteria  (see  Part  II.,  Chapter  II.).  The  develop- 
ment of  acids  may  be  detected  after  the  addition  of  2  per  cent, 
of  rosolic  acid  solution  (0.5  per  cent,  solution  in  alcohol); 
alakaline  solutions  give  a  clear  rose-color  which  disappears  in 
the  presence  of  acids. 

Blood-serum. — The  blood  of  the  ox  or  cow  may  be  obtained 
easily  at  the  abattoir.  It  should  be  collected  in  a  clean  jar. 
When  it  has  coagulated,  the  clot  should  be  separated  from  the 
side  of  the  jar  with  a  glass  rod.  It  may  be  left  on  the  ice  for 
from  twenty-four  to  forty-eight  hours.  At  the  end  of  that  time 
the  serum  will  have  separated  from  the  clot  and  may  be  drawn 
off  with  a  siphon  or  pipette  into  tubes.  The  tubes  containing 
the  serum  should  be  placed  in  a  slanting  position,  as  nearly 


74  MANUAL    OF    BACTERIOLOGY. 

horizontal  as  possible  without  bringing  the  serum  in  contact 
with  the  cotton  plug,  and  while  in  this  position  they  should  be 
heated  gradually  up  to  65-68°  C.  This  is  best  done  in  a 
specially  constructed  Koch  serum  coagulator,  but  it  may  also 
be  done  as  advised  by  Councilman  and  Mallory,  in  the  hot-air 
sterilizer  at  a  temperature  below  the  boiling-point  or  it  may  be 
accomplished  by  means  of  a  water  bath  or  in  the  Arnold 
sterilizer  or  in  other  ways.  The  more  gradually  the  heat  is 
raised  and  the  lower  the  temperature  at  which  the  serum  is 
coagulated  the  more  transparent  it  remains.  After  coagulation 
the  tubes  of  serum  maybe  sterilized  in  the  Arnold  sterilizer  on 
three  successive  days,  or  they  may  be  sterilized  in  the  autoclave 
at  110°  at  one  time.  Sterilization  of  blood-serum  in  the  auto- 
clave is  not  recommended  by  some  authors.*  Blood-serum 
may  be  sterilized  in  the  special  form  of  sterilizer  devised  for  it. 
A  clear  fluid  blood-serum  may  be  obtained  by  sterilization  at  a 
temperature  of  56  to  58°  C.  for  one  hour,  on  each  of  six  days. 
Opaque,  coagulated  blood-serum  has  most  of  the  advantages  of 
the  clear  medium.  Blood-serum  may  be  secured  from  small 
animals  by  collecting  blood  directly  from  the  vessels,  using 
very  great  care  to  obtain  the  blood  in  a  sterile  condition;  and 
the  serum  may  be  separated  and  stored  in  a  fluid  state.  Human 
blood-serum  is  sometimes  obtained  from  the  placental  blood, 
sometimes  from  serous  pleural  transudates  or  from  hydrocele 
fluid.  The  preservation  of  blood-serum  is  sometimes  accom- 
plished with  chloroform,  of  which  i  per  cent,  is  to  be  added  to 
the  medium;  in  this  manner  the  serum  may  be  preserved  for  a 
long  time.  It  may  be  divided  into  tubes,  solidified  and  steri- 
lized as  required;  the  chloroform  is  driven  off  by  the  heat,  in 
sterilizing,  but  it  must  be  heated  to  at  least  68°  C.  Blood- 
serum  media  which  are  sterilized  at  low  temperatures  should 
be  tested  for  twenty-four  hours  in  the  incubator  to  prove  that 

*Park.     Pathogenic  Bacteria  and  Protozoa.     New  York  and  Phila.,  1905, 
P-  51- 


CULTURE-MEDIA.  75 

sterilization  has  been  effective;  if  it  has  not,  development  of  the 
contaminating  bacteria  will  take  place  and  be  visible  to  the  eye. 

It  will  be  impossible  to  do  more  than  merely  mention  some 
of  the  most  important  of  the  other  culture-media. 

Loffler's  blood-serum  consists  of  one  part  of  bouillon  con- 
taining i  per  cent,  of  glucose  and  three  parts  of  blood-serum. 
It  is  sterilized  like  ordinary  blood-serum.  It  is  used  largely 
for  the  cultivation  of  the  bacillus  of  diphtheria. 

Blood-serum-agar  is  a  medium  made  with  considerable  dif- 
ficulty, but  when  made  with  human  blood-serum  very  valuable 
for  the  cultivation  of  the  gonococcus.  One  part  of  placental 
blood-serum,  or  pleuritic  serum,  or  hydrocele  fluid,  is  mixed 
with  one  to  two  parts  of  nutrient  agar  in  the  fluid  condition. 
It  must  be  divided  into  tubes  before  solidification.  It  should  be 
solidified  in  a  slanting  position,  and  sterilized  at  58°  C.  so  as 
1  not  to  coagulate  the  blood-serum.  At  this  temperature  it  is 
necessary  to  sterilize  for  several  successive  days  (see  page  74) 
and  it  should  be  tested  in  the  incubator  for  sterility.  The 
nutrient  agar  in  this  case  should  contain  2  per  cent,  of  dry  agar. 

Another  expedient  has  also  been  to  smear  a  little  blood,  drawn  from  a  puncture 
made  by  a  sterile  needle  in  the  carefully  cleaned  finger,  over  the  surface  of  a 
tube  of  nutrient  agar — blood-agar — used  for  cultivating  the  bacillus  of  influenza. 
In  this  case  the  finger  from  which  the  blood  is  drawn  is  scrubbed  with  soap  and 
water,  soaked  with  T^  ^  corrosive  sublimate,  and  finally  washed  with  alcohol. 
Small  quantities  of  blood  may  be  drawn  from  a  vein  in  the  ear  of  a  rabbit  (see 
page  103)  with  a  sterile  hypodermic  syrince,  and  is  quickly  divided  among  three 
or  four  tubes  of  agar,  melted  in  the  upper  third;  slant  the  tubes  while  cooling. 
To  make  a  large  amount  of  blood-agar,  bleed  a  rabbit  from  the  carotid  artery 
into  a  sterile  flask  containing  pieces  of  sterile  glass  tubing;  shake  the  flask  con- 
stantly; divide  the  defibrinated  blood  among  tubes  containing  sterile  nutrient 
agar;  slant  the  tubes  while  cooling.  Use  one  part  of  blood  to  about  two  of  agar. 
Great  care  must  be  used  not  to  contaminate  the  blood  as  it  is  drawn,  and  it  is 
best  to  test  it  in  the  incubator  for  sterility.  The  tubes  when  completed  should 
stand  some  days  before  using,  so  that  contaminating  bacteria  if  present  may 
grow  in  the  interval  and  permit  such  tubes  to  be  discarded. 

Guarnieri's  medium  consists  of  a  mixture  of  gelatin  and  "agar. 

Eggs  in  their  shells  may  be  used  after  sterilization  by  steam,  which  of  course 


y6  MANUAL    OF    BACTERIOLOGY. 

coagulates  the  albumen.  The  egg  is  easily  inoculated  through  a  small  opening 
made  with  a  heated  needle,  which  may  be  closed  afterward  with  collodion. 
Egg-albumen  has  been  used  as  a  constituent  of  various  media.  Dorset*  has 
found  that  eggs  furnish  an  excellent  culture-medium  for  tubercle  bacilli.  The 
yolk  and  trie  white  are  mixed,  poured  into  tubes,  slanted,  coagulated,  and 
sterilized.  Just  before  using  pour  into  the  tube  a  few  drops  of  sterile  distilled 
water  to  moisten  the  medium.  This  is  a  most  valuable  addition  to  the  technic. 
Bread-paste  (finely-divided  dry  bread,  mixed  with  water"  and  sterilized)  is 
used  for  the  cultivation  of  moulds.  Sabouraud  recommends  the  following  for 
the  cultivation  of  the  trichophyton  fungus: 

Peptone 5.0  grams. 

Maltose       ' .       3-8  grams. 

Agar        1.3  grams. 

Water      100.0  c.c. 

Test-tubes. — Bacteria  are  generally  cultivated  in  test-tubes. 
A  convenient  size  is  one  f  of  an  inch  in  diameter  and  5  inches 
in  length.  The  tubes  should  be  of  a  heavier  glass  than  in  those 
used  for  ordinary  chemical  work.  The  New  York  Board  of 
Health,  and  some  others,  use  a  tube  three  inches  in  length  with- 
out a  flange  for  the  cultivation  of  the  diphtheria  bacillus  on 
Loffler's  blood-serum  mixture.  Test-tubes  should  be  thor- 
oughly cleaned  with  a  swab  before  using;  they  should  be  boiled 
with  washing-soda,  rinsed,  rilled  with  hydrochloric  acid  solu- 
tion, rinsed  and  inverted  to  drain  away  the  fluid. 

Plugs  of  raw  cotton  or  cotton  batting  are  employed  as  stop- 
pers. Some  prefer  absorbent  cotton,  but  it  is  likely  to  become 
soggy  after  exposure  to  steam.  The  plug  should  fit  smoothly; 
creases  and  cracks  around  the  edges  are  to  be  avoided.  The 
plug  should  be  tight  enough  to  sustain  the  weight  of  the  tube 
when  held  by  the  plug.  These  plugs  prevent  bacteria  from 
entering  or  leaving  the  tubes. 

Sterilization  of  Test-tubes. — The  tubes  are  to  be  sterilized 
in  a  hot-air  sterilizer  for  one  hour,  at  a  temperature  of  150° 
C.  They  may  be  left  in  until  the  cotton  acquires  a  light-brown 
color,  but  it  should  not  be  burned.  If  the  plugs  touch  the  sides 
of  the  sterilizer  or  lie  against  the  bottom  they  may  be  scorched. 

^American  Medicine.     April  5,  1902. 


CULTURE-MEDIA. 


77 


The  necessity  for  sterilization  of  the  tubes  before  filling  them 
with  the  medium  has  been  questioned,  and  it  is  probably  un- 
necessary as  far  as  the  preservation  of  the  culture-medium  is 
concerned,  but  it  will  be  found  that  the  cotton  plugs  fit  much 
better  after  sterilization  with  dry  heat.  During  this  and  subse- 
quent sterilizations  the  tubes  are  held  in  a  wire  basket. 

Filling  of  the  Tubes. — A  special  funnel  closed  with  a  stop- 
cock for  filling  tubes  with  liquefied  media  is  often  recom- 
mended. They  may  readily  be  filled  with  an  ordinary  funnel 
of  small  size.  During  the  filling,  the  neck  of  the  test-tube 
where  it  comes  in  contact  with  the  cotton 
must  not  be  wet  with  the  medium.  Or- 
dinarily about  7  to  10  c.c.  are  placed  in 
a  test-tube.  For  Esmarch's  roll-tubes  a 
somewhat  smaller  quantity  is  desirable. 

The  sterilization  of  tubes  containing 
culture-media  is  always  done  by  steam 
and  has  been  sufficiently  described.  It 
is  to  be  remembered  that  the  solidifying 
power  of  gelatin  is  impaired  by  too  pro- 
longed heating,  while  heating  is  less 
likely  to  damage  other  culture-media. 

The  media  which  are  sterilized  at  a  low  temperature  (70°  C). 
should  be  tested  for  two  days  in  the  incubator  to  determine 
whether  sterilization  has  been  effective.  It  is  the  universal 
experience  in  bacteriological  laboratories  that  occasionally 
culture-media  will  become  contaminated  with  extremely  resist- 
ant spores  which  fail  to  be  sterilized  by  the  ordinary '  pro- 
cesses, an  occurrence  which  causes  great  annoyance  and  calls 
for  the  exercise  of  much  patience.  Sometimes,  also,  moulds 
attach  themselves  to  the  plugs,  especially  if  they  are  moist, 
and  send  their  filaments  down  through  the  cotton;  finally, 
having  reached  the  lower  edge  of  the  cotton,  their  spores  may 
fall  upon  the  medium,  grow  there'  and  ruin  it. 


FIG.  18.— Wire  basket 
for  test-tubes. 


CHAPTER  IV. 
THE  CULTIVATION  OF  BACTERIA. 

Inoculation  of  the  Tubes. — The  air  of  the  laboratory 
should  be  as  quiet  as  possible,  in  order  to  lessen  the  chances 
of  contamination  by  bacteria  clinging  to  particles  of  dust. 
Spores  are  blown  from  the  surfaces  of  moulds  like  thistle- 
down, and  are  constantly  being  wafted  about  in  the  air  where 
there  are  draughts.  After  the  colonies  are  obtained  on  the 
plate  or  in  the  Esmarch  tube  a  pure  culture  is  obtained  by  trans- 
ferring a  minute  amount  of  the  growth  from  a  colony  over  into 
a  test-tube  containing  the  sterile  culture  medium.  The  transfer 
is  effected  by  means  of  a  straight  platinum  wire,  or  with  a  plati- 
num wire  loop.  The  platinum  is  to  be  sterilized  by  heating 
to  a  red  heat  in  the  Bunsen  flame  or  with  an  alcohol  lamp  before 
using,  and  then  allowed  to  cool.  It  is  also  to  be  heated  red- 
hot  after  using.  The  plug  of  the  test-tube  is  withdrawn, 
twisting  is  slightly,  taking  it  between  the  third  and  fourth 
ringers  of  the  left  hand,  with  the  part  that  projects  into  the  tube 
pointing  toward  the  back  of  the  hand.  It  must  not  be  allowed 
to  touch  any  object  while  out  of  the  tube.  The  upper  inch 
or  two  of  the  tube  should  be  passed  through  the  flame  in  order 
to  destroy  any  bacteria  which  may  settle  on  it  from  the  air  while 
the  plug  is  out.  If  any  of  the  cotton  adheres  to  the  in- 
side of  the  tube  it  should  be  removed  with  sterilized  for- 
ceps, while  the  neck  of  the  tube  touches  the  flame,  so  that 
the  threads  of  cotton  may  be  burned  and  not  fly  into  the 
air  of  the  room.  The  tube  should  be  held  as  nearly  hori- 
zontal as  possible.  The  tube  should  be  held  in  the  left 
hand  between  the  thumb  and  forefinger,  resting  upon  the 

78 


THE    CULTIVATION   OF   BACTERIA.  79 

palm,  with  the  mouth  pointing  upward  and  to  the  right. 
When  two  tubes  are  being  used  at  the  same  time,  as 
is  often  necessary,  they  are  placed  side  by  side  between  the 
thumb  and  forefinger  of  the  left  hand.  The  two  plugs  are  held 
between  the  second  and  third  and  the  third  and  fourth  fingers 
of  the  left  hand,  respectively.  Inoculation  from  one  tube  to  the 
other  is  effected  by  touching  the  tip  of  the  platinum  wire  to  the 
material  to  be  transferred  in  the  one  tube,  and  introducing  it 
into  the  culture-medium  contained  in  the  other  tube.  When 
the  needle  is  intrcduced  into  or  removed  from  either  tube  it 
should  not  touch  the  side  of  the  tube  at  any  point,  but  be 


FIG.  19. — Manner  of  holding  tubes. 

brought  in  contact  only  with  the  medium  to  be  inoculated. 
After  inoculation  of  the  tube  has  been  effected  the  wire  must 
be  heated  to  a  red  heat  in  the  flame,  the  mouths  of  the  tubes 
passed  through  the  flame,  and  the  plugs  returned  to  their 
respective  tubes.  When  the  wire  to  be  sterilized  is  wet  it 
should  be  approached  to  the  flame  gradually,  so  as  to  dry  the 
material  on  it,  in  order  to  avoid  " sputtering"  (see  page  22). 
It  is  well  from  the  start  to  train  one's  self  to  sterilize  the  platinum 
wire  every  time  it  is  taken  from  the  table  and  before  it  is  laid 
down  again.  'The  platinum  loop  is  used  in  place  of  the 
straight  wire  when  it  is  desired  to  transfer  larger  amounts  of 
material  than  can  be  done  at  one  time  on  a  straight  wire, 


So  MANUAL    OF    BACTERIOLOGY. 

and  is  employed  in  case  of  material  containing  a  small  number 
of  bacteria. 

When  a  tube  of  gelatin  is  to  be  inoculated  the  wire  is 
usually  introduced  into  the  medium  vertically,  " stab-culture;" 
when  a  medium  with  a  slanted  surface  is  employed,  as  agar, 


FIG.  20. — Stab  culture.     A  rubber  FIG.    21. — Smear    culture.       This 

stopper    may    be    use      to    prevent         tube  shows  the  rubber  cap  used  to 
drying,  see  page  86.  prevent  drying. 

potato  or  blood-serum,  the  needle  should  lightly  streak  the 
surface,  " smear  culture"  (Figs.  20  and  21). 

The  safety  and  success  of  this  method  of  inoculation  depend 
upon  a  principle  which  has  been  established  by  long  and  re- 
peated observation, — namely,  that  bacteria  do  not  of  them- 
selves leave  a  moist  surface,  although  they  may  be  readily 
shaken  off  as  would  appear  from  Flugge's*  results.  Neverthe- 

*Zeitschri/t.  /.  Hygiene  Bd.  25,  1897,  p.  179. 


THE    CULTIVATION   OF   BACTERIA.  8 1 

less,  they  will  not  rise  from  the  surface  of  the  moist  culture- 
medium,  nor  drop  from  the  needle  during  its  transit,  if  proper 
care  be  exercised.  They  may  be  thrown  into  the  air  if  the 
needle  be  allowed  to  sputter  in  the  flame,  or  is  roughly  shaken 
about. 

It  must  be  remembered  also  that  such  organisms  as  moulds  develop  spores 
which  are  formed  on  filaments  elevated  above  the  surface  of  the  medium  and  are 
easily  detached. 

If,  by  any  accident,  drops  of  infectious  material  should  fall 
upon  a  surface  like  the  table,  they  should  be  covered  at  once 
with  bichloride  of  mercury  solution  i-iooo.  A  good  way  is 
to  cover  the  spot  with  a  piece  of  blotting-paper  wet  with  the 
solution,  place  a  bell-jar  over  it  and  leave  for  several  hours. 
If  infectious  material  should  reach  the  hands  or  clothing,  they 
should  be  thoroughly  soaked  in  the  bichloride  solution.  When 
working  with  pathogenic  bacteria  it  is  well  to  wash  the 
hands  in  this  solution  and  with  soap  and  water,  as  a  routine 
procedure,  before  leaving  the  laboratory. 

To  maintain  their  vitality  bacteria  need  to  be  transplanted 
from  time  to  time  from  the  medium  in  which  they  are  culti- 
vated to  fresh  medium;  the  frequency  with  which  this  trans- 
plantation is  necessary  varies  greatly  with  different  species. 
When  transplanted  from  one  tube  to  another  the  bacteria  should 
be  examined  with  the  microscope  both  in  the  original  tube  and  in 
the  resulting  growth  in  order  to  check  the  purity  of  the  culture. 
Bacteria  differ  greatly  in  the  ease  with  which  they  may 
be  cultivated  artificially.  Many  of  them  grow  on  culture- 
media  with  difficulty  when  first  taken  from  the  animal  body, 
or  when  first  transplanted  from  one  sort  of  medium  to  a  differ- 
ent one.  But  they  become  accustomed  to  such  changes  of 
conditions  and  frequently  may  be  propagated  easily  on  the  new 
medium.  This  is  especially  true  of  bacillus  tuberculosis. 

From  what  has  just  been  said,  it  is  evident  that  some  bacteria 
flourish  better  on  one  culture-medium  than  on  another.  The 

' 


82  MANUAL   OF    BACTERIOLOGY. 

bacillus  tuberculosis  grows  best  on  boiled  egg,  blood-serum 
and  glycerin- agar;  the  bacillus  of  diphtheria  grows  best  on 
Lofflers  blood-serum;  the  gonococcus  on  human  serum-agar. 
The  virulence  of  most  pathogenic  bacteria  becomes  dimin- 
ished after  prolonged  cultivation  upon  artificial  media.  Some- 
times the  virulence  is  lost  very  quickly, — for  example,  the 
streptococcus  pyogenes  and  micrococcus  lanceolatus  of  pneu- 
monia. These  organisms  are  often  very  strongly  patho- 
genic for  experiment  animals,  when  inoculated  directly  from 
one  animal  to  another,  but  frequently  lose  all  pathogenic 
power,  even  on  one  transfer  to  artificial  media. 

Incubators. 

High-temperature  Incubator — Many  bacteria  flourish  best  at 
a  temperature  about  that  of  the  human  body,  38°  C.  Some 
species  will  grow  only  at  this  temperature.  The  pathogenic 
bacteria  in  particular,  for  the  most  part,  thrive  best  at  a 
point  near  the  body  temperature,  and  are  consequently  best 
studied  for  many  purposes  when  grown  in  an  incubator, 
regulated  for  this  temperature. 

The  high-temperature  incubator  used  in  laboratories  con- 
sists essentially  of  a  box  made  of  copper,  having  double  walls, 
the  space  between  the  two  being  filled  with  water.  The  outer 
surface  is  covered  with  some  non-conductor  of  heat,  such  as 
felt,  asbestos  or  linoleum.  At  one  side  is  a  door,  which  is  also 
double.  The  inner  door  is  of  glass,  the  outer  door  is  of  copper 
covered  with  asbestos.  At  one  side  is  a  gauge  which  indi- 
cates the  level  at  which  the  water  stands  in  the  water-jacket. 
The  roof  is  perforated  with  several  holes,  some  of  which  per- 
mit the  circulation  of  the  air  in  the  air-chamber  inside  the 
box;  some  of  them  enter  the  water-jacket.  A  thermometer 
passes  through  one  of  these  holes  into  the  interior  of  the  air- 
chamber,  and  often  another  into  the  water  standing  in  the 
water-jacket.  A  gas-regulator  passes  through  another  hole, 


THE    CULTIVATION   OF    BACTERIA.  83 

and  is  immersed  in  the  water  standing  in  the  water-jacket. 
There  are  various  forms  of  gas-regulators  more  or  less 
complicated.  In  general  they  consist  of  a  tube  containing 
mercury;  into  this  tube  are  two  openings,  one  for  the  entrance 


FIG.  22. — Incubator. 


and  the  other  for  the  exit  of  gas.  The  gas  enters  through  a 
small  tube,  which  is  cut  off  diagonally  at  the  bottom,  and  which 
projects  into  the  surface  of  the  mercury.  Heating  the  water 
in  the  water-jacket  causes  expansion  of  the  mercury,  which 


84 


MANUAL   OF    BACTERIOLOGY. 


rises,  and,  little  by  little,  cuts  off  the  inflow  of  gas  through 
this  tube.  The  flow  is  never  completely  cut  off,  as  there  is  a 
capillary  opening  in  the  tube  considerably  above  any  point  to 
which  the  mercury  could  possibly  rise,  which  will  always  allow 
the  flow  of  a  small  quantity  of  gas  (Fig.  25,  b}.  This  diagram 
also  shows  a  modification  of  the  simple  form  of  regulator,  in 


; 


FIG.  23. — Reichert's  Gas- 
regulator. 


FIG.  24. — Mercurial  Gas-regulator. 
a.  Chamber  containing  volatile  hy- 
drocarbon, b.  Capillary  opening. 


the  shape  of  a  partition  which  divides  off  a  lower  chamber, 
which  contains  mercury  and  is  connected  with  the  upper  part 
by  a  glass  tube.  The  purpose  is  to  make  use  of  the  elastic 
properties  of  some  volatile  fluid,  like  ether,  which  floats  on  the 
surface  of  the  mercury  at  a. 

A  most  satisfactory  gas  regulator  is  that  of   Roux.     It  is 
constructed  entirely  of  metal,  and  its  operation  is  due  to  the 


THE    CULTIVATION    OF    BACTERIA. 


unequal  expansion  and  contraction  of  two  metals  which  are 
riveted  together.  Fig.  25  shows  this  regulator.  The  gas 
passes  in  at  e  and  passes  out  at  d.  The  amount  of  gas  pass- 
ing through  is  regulated  by  a  piston  on  the  end  of  the  set 
screw  inside  the  tube  from  which  the  outlet  tube  branches  off. 
This  piston  moves  in  or  out  according 
to  the  changes  of  temperature  of  the 
water  jacket  of  the  incubator  into 
which  the  stem  (/)  of  the  regulator  is 
inserted.  This  stem  is  finestrated  and 
has  the  riveted  metallic  strips  running 
down  in  it.  These  strips  are  pivoted 
at  the  collar,  g. 

The  gas  coming  from  the  gas-regu- 
lator passes  to  a  Bunsen  burner,  which 
stands  underneath  the  incubator. 
This  burner  should  have  some  kind 
of  automatic  device  for  cutting  off  the 
flow  of  gas  in  case  it  becomes  acci- 
dentally extinguished  by  a  sudden 
draught  of  air  or  from  any  other 
cause.  The  automatic  burner  in- 
vented by  Koch  is  an  ingenious, 
simple  and  effective  device  (Fig.  26). 
The  coils  of  metal  seen  on  each  side 
at  the  top  of  the  burner  are  so 
arranged  that  when  they  expand 
they  turn  the  disk  below  so  as  to 

support  the  arm  coming  from  the  stop-cock;  when  they 
cool  they  turn  the  disk  in  the  opposite  direction,  and 
allow  the  arm  to  fall  and  cut  off  the  gas.  Some  incon- 
venience will  arise  from  irregularities  in  the-  flow  of  gas 
from  the  main  supply-pipe.  Any  incubator  will  vary  a  little 
from  such  causes.  In  the  experience  of  the  writer,  natural 


FIG.  25. — Roux  Bimetallic 
Gas  Regulator,  a,  Set  screw; 
b,  Screw  collar;  c,  Clamp; 
d,  Outlet  for  gas;  e,  Inlet  for 
gas. 


86 


MANUAL    OF    BACTERIOLOGY. 


gas  is  of  such  variable  pressure  as  to  be  entirely  useless. 
Fluctuations  of  the  temperature  within  the  incubator  depend 
very  largely  upon  the  external  temperature.  Therefore,  the 
incubator  should,  as  far  as  is  practicable,  be  protected  from 
sudden  draughts  of  cold  air  and  should  be  kept  in  a  room 
having  as  equable  a  tempera  ture  as  possible.  In  large 
laboratories  it  is  often  found  convenient  to  use  the  whole  of 
a  small  room  as  an  incubator,  heating  it  by  a  gas  stove,  to 
which  a  gas  regulator  may  be  applied. 

Culture-tubes  which  are  being  kept  in  the  incubator  are 
likely  to  become  dry  if  their  stay  is  pro- 
longed. In  such  cases  they  should  be 
covered  with  rubber  caps,  tin-foil,  seal- 
ing-wax, paraffin  or  some  other  device  to 
prevent  evaporation.  If  rubber  caps  are 
used,  they  should  be  left  in  i-iooo 
bichloride  of  mercury  solution  for  an 
hour,  and  the  cotton  plugs  should  be 
singed  in  the  flame  before  putting  them 
on.  (Fig.  21.)  The  wiiter  prefers  rubber 
stoppers,  which  may  be  boiled  and  stored 
in  bichloride  of  mercury  solution.  In 
using  stoppers  in  this  way  the  cotton  plug  is  cut  even  with  the 
mouth  of  the  tube,  the  top  singed  in  the  flame,  and  the  plug 
shoved  down  into  the  tube  for  about  i  cm.  The  rubber 
stopper  is  then  inserted  (Fig.  20). 

Low -temperature  Incubator. — An  incubator  regulated  for  so- 
called  "room  temperature"  is  very  desirable  for  the  cultivation 
of  bacteria  upon  gelatin  and  for  the  bacteriological  analysis 
of  water.  In  our  climate  the  temperature  of  the  rooms  of  the 
laboratory  often  reaches  a  point  at  which  gelatin  melts,  and 
for  this  reason  in  a  low-temperature  incubator  provision  has  to 
be  made  for  cooling  when  the  room  temperature  is  too  high  as 
well  as  for  heating  when  it  is  too  low. 


FIG.  26.  Koch  Auto- 
matic Gas-burner. 


THE    CULTIVATION   OF    BACTERIA.  87 

A  form  of  incubator  devised  by  Rogers*  for  this  purpose 
consists  of  a  refrigerator  or  of  a  specially  constructed  chamber 
heated  by  electricity  and  controlled  by  an  electric  thermoregu- 
lator.  Below  is  given  a  description  of  an  incubator  constructed 
according  to  Rogers'  plans.  This  incubator  has  been  in  use 
for  some  time  and  has  given  entire  satisfaction  since  the  pre- 
cautions noted  below  were  followed.  There  would  appear 
no  reason  why  this  incubator  should  not  be  employed  for  high 
temperatures  as  well  for  low,  but  so  far  it  has  been  run  at  22°  C. 
The  temperature  has  kept  very  constant.  The  incubator 
consists  of  a  refrigerator,  30  inches  high,  24  inches  wide,  18 
inches  from  front  to  back,  all  outside  measurements.  Instead 
of  the  ordinary  drip  pipe,  there  is  a  coil  of  i-inch  galvanized 
iron  pipe  run  down  the  back  of  the  cooling  chamber  attached 
water-tight  to  the  ice  tank.  From  the  bottom  of  the  cooling 
chamber  the  coil  runs  up  perpendicularly  nearly  to  the 
bottom  of  the  ice  compartment,  and  then  runs  horizontally 
through  the  wall  of  the  refrigerator.  A  bracket  on  the  outside 
supports  a  drip-pan.  A  thermometer  encased  in  a  finestrated 
metal  jacket  is  inserted  about  half  way  up  on  one  side.  A 
lump  of  ice,  about  50  pounds,  placed  in  the  ice  compartment 
serves  to  keep  the  temperature  sufficiently  cool.  In  summer 
doubtless  more  ice  will  be  required. 

For  heating,  an  ordinary,  i6-candle-power  electric  bulb  is 
used,  and  the  electricity  is  obtained  from  the  public  supply. 
The  wire  is  run  through  one  of  the  walls,  and  a  part  of  the 
current  is  made  to  operate  a  horse-shoe  magnet,  and  another 
part  is  conducted  through  the  lamp  used  for  heating. 

The  accompanying  diagram,  (Fig.  27),  will  serve  to  show 
the  arrangement. 

A  telegraph  key  is  used  to  supply  the  horse-shoe  magnet 

*L.  A.  Rogers.  On  electrically  controlled  low  temperature  incubators. 
Centralblatt  fur  Bakteriologie,  etc.  Bd.  XV.  Abt.  II.  Double  No.  7-8.  pp . 
236-239.  Sept.  23,  1905. 


88 


MANUAL    OF    BACTERIOLOGY. 


which  is  inserted  in  the  heating  circuit  in  such  a  way  that 
when  the  armature  is  attracted  toward  the  magnet  the  circuit 
is  completed  and  the  lamp  is  consequently  lighted.  The  part 
of  the  current,  a,  supplying  the  magnet  first  passes  through 
a  small  lamp  and  through  two  resistance  coils  so  as  to  reduce 
the  pressure.  It  then  passes  through  the  magnet,  and  is  con- 
tinued on  to  the  set-screw,  b,  which  is  so  placed  that  when  the 
thermoregulator  comes  in  contact  with  it  the  current  is  com- 
plete. The  regulator  consists  of  a  strip  of  hard  rubber  and  a 


-Kara/ Rubber: 
Thermo rea/u 'later 

FIG.  27. 


'strip  of  brass  riveted  together.  One  end  is  fixed,  while  the 
other  is  free,  and  when  it  is  heated  it  tends  to  bend  toward 
the  metal  side,  when  it  cools  it  bends  toward  the  rubber. 
The  brass  strip  is  15  inches  long,  J  inch  thick,  and  J  inch  wide; 
the  rubber  strip  is  \  inch  thick,  \  inch  wide,  and  a  little  less 
than  15  inches  long.  In  the  diagram  the  end  is  fixed  at  d 
and  is  free  at  b.  When  it  is  heated,  the  free  end  travels  away 
from  the  set-screw  at  b;  when  it  cools,  if  moves  toward  the  set- 
screw.  Rogers  also  recommends  a  regulator  made  of  invar  and 


THE    CULTIVATION    OF   BACTERIA.  89 

brass  instead  of  hard  rubber  and  brass.  Where  invar  is  used 
instead  of  the  hard  rubber  the  dimensions  for  the  two  metals 
are  the  same  as  those  given  for  the  brass  strip  in  the  hard 
rubber-brass  regulator  just  described.  As  is  evident  from  the 
description,  the  circuit  controlling  the  magnet  is  closed  when- 
ever the  free  end  of  the  regulator  comes  in  contact  with  the 
set  screw  at  b.  When  this  circuit  is  closed  the  magnet  attracts 
the  armature,  and  the  heating  circuit  is  closed  by  the  contact 
formed  at  c  between  the  armature  and  the  set-screw.  In  the 
diagram  this  point  of  contact  is  put  to  one  side  for  the  sake 
of  clearness,  but  as  a  matter  of  fact  in  the  instrument  in  use 
the  set-screw  is  above  and  between  the  ends  of  the  horse- 
shoe magnet,  and  comes  in  contact  with  the  armature  which  is 
extended  upward  in  the  shape  of  a  tongue.  From  the  descrip- 
tion just  given  it  will  be  noted  that  the  thermoregulator  does 
not  regulate  the  heating  directly,  but  indirectly  through  the 
electro-magnet.* 

Certain  precautions  have  been  found  necessary  in  practice 
in  order  to  obtain  satisfactory  results  with  this  incubator.  The 
set-screw  against  which  the  armature  strikes  at  c  should  be  so 
set  that  the  armature  does  not  come  in  contact  with  the  mag- 
net. In  the  apparatus  described  above  there  is  a  space  of  ab- 
out J  inch  between  the  armature  and  the  magnet  when  contact 
takes  place  between  the  set-screw  and  the  armature.  If  the 
set-screw  does  not  project  far  enough  to  prevent  the  armature 
from  coming  in  contact  with  the  magnet  the  armature  is  apt 
to  stick  to  the  magnet  even  after  the  current  is  broken  at  6, 
and  when  this  is  the  case  of  course  the  lamp  remains  lighted, 
and  the  temperature  may  run  up  too  high.  This  sticking  of 
the  armature  to  the  magnet  is  said  to  be  due  to  the  residual 
magnetism  left  in  the  core  of  the  magnet.  When  the  current 

*  Recently  the  regulator  has  been  connected  directly  with  the  current  pass- 
ing through  the  heating  lamp  without  the  intervention  of  the  horse  shoe  mag- 
net, connecting  the  wires  e  and  /  directly  with  d  and  b. 


9o 


MANUAL    OF    BACTERIOLOGY. 


passing  through  the  magnet  is  broken  by  the  excursion  of  the 
end  of  the  thermoregulator  away  from  the  set-screw  at  b, 
the  armature  is  pulled  away  from  the  magnet  by  a  coiled  spring. 
Another  important  precaution  is  that  the  points  at  which  con- 
tact is  made  and  broken,  b  and  c,  should  be  tipped  with  plati- 
num. A  small  piece  of  platinum 
wire  inserted  into  the  ends  of  the 
set-screws  and  a  few  square 
centimeters  of  platinum  foil 
riveted  to  the  opposite  point  of 
contact  meets  the  requirements. 
If  platinum  is  not  used  at  these 
points  oxydation  takes  place  and 
prevents  contact.  The  set-screw 
at  b  is  set  by  experiment  for 
the  temperature  desired.  The 
further  the  point  of  the  set- 
screw  projects  toward  the  free 
arm  of  the  regulator,  the  higher 
the  temperature  maintained. 

CULTIVATION  OF  ANAEROBIC 
BACTERIA. 


FIG.  28. — Arrangement  of  tubes 
for  cultivation  of  anaerobes  by 
Buchner's  method. 


The  cultivation  of  anaerobic 
bacteria  is  done  best  in  a  medium 
containing  i  to  2  per  cent,  of 
dextrose.  The  tube  should  con- 
tain a  large  quantity  of  the 

culture-medium.  Just  before  using,  the  medium  should  be 
boiled  for  a  few  minutes.  Inoculate  the  tube  after  cooling, 
but  while  the  medium  is  fluid.  Anaerobes  may  be  cultivated 
in  the  closed  arm  of  the  fermentation-tube  (see  Fig.  $f,  but 
the  opening  between  the  two  arms  of  the  tube  must  be  small. 
Buchner's  Method  jor  the  Cultivation  of  Anaerobes. — Into  a 


THE   CULTIVATION   OF   BACTERIA. 


9T 


bottle  or  tube  which  can  be  tightly  stoppered,  pour  10  c.c.  of 
a  6  per  cent,  solution  of  sodium  or  potassium  hydroxide,  for 
each  100  c.c.  of  air  contained  in  the  jar.  Add  one  gram  of 
pyrogallic  acid  for  each  10  c.c.  of  solution.  The  culture-tube 
is  placed  inside  of  the  larger  bottle  or  tube,  supported  above 
the  bottom,  and  the  stopper,  smeared  with  paraffin,  is  inserted. 
The  mixture  of  pyrogallic  acid  and 
potassium  hydroxide  possesses  the  pro- 
perty of  absorbing  oxygen. 

Wright's  Modification  of  Buchner's 
Method. — The  tube  of  culture-medium 
is  to  be  plugged  with  absorbent  cotton, 
using  a  plug  of  large  size.  The  culture- 
medium  is  inoculated  in  the  usual  way. 
The  plug  is  cut  off  close  to  the  neck  of 
the  tube,  and  is  then  pushed  into  the 
tube  about  i  centimeter.  Now  allow  a 
watery  solution  of  pyrogallic  acid  to  run 
into  the  plug,  and  then  a  watery  solution 
of  sodium  or  potassium  hydroxide. 
Close  quickly  and  tightly  with  a  rubber 
stopper.  Wright  recommends  that  the 
first  solution  be  freshly  made  and  consist 
of  about  equal  volumes  of  pyrogallic 
acid  and  water,  and  that  the  second 
solution  contain  i  part  of  sodium  hy- 
droxide and  2  parts  of  water.  With  6-inch  test-tubes,  f-inch 
diameter,  the  amounts  advised  are— J  c.c.  solution  of  pyrogallic 
acid,  i  c.c.  solution  of  sodium  hydroxide. 

Cultivation  of  Anaerobic  Bacteria  under  Hydrogen:  Method  of  Liborius 
Modified  by  Frankel. — A  test-tube  containing  a  large  amount  of  the  liquefied 
culture-medium  is  closed  with  a  sterilized  rubber  stopper,  through  which  pass 
two  sterilized  glass  tubes,  bent  above  the  stopper  at  a  right  angle.  One  of  these 
tubes  is  cut  off  just  underneath  the  stopper,  and  the  other  is  long  enough  to 
project  nearly  to  the  bottom  of  the  culture-tube.  The  horizontal  projecting 


FIG.  29. — Cultivation  of 
anaerobes  by  Flankers 
method. 


MANUAL   OF    BACTERIOLOGY. 


parts  are  drawn  to  a  small  caliber  at  some  point,  although  not  quite  closed,  to 
facilitate  sealing  later  on.  Through  the  longer  of  these  tubes  hydrogen  gas  is 
passed  until  the  atmosphere  inside  of  the  culture-tube  is  pure  hydrogen,  entirely 
free  from  mixture  with  air.  The  horizontal  parts  of  the  small  glass  tubes 
projecting  from  the  stopper  are  then  sealed  in  the  flame  at  the  places  where  they 
were  previously  drawn  out  to  a  small  caliber,  and  the  tubes  are  thus  closed. 
(Fig.  27.) 

The  stopper  should  be  surrounded  with  melted  paraffin.  A  tube  prepared 
according  to  this  plan  may,  if  desired,  be  converted  into  an  Esmarch  roll-tube. 
The  hydrogen  is  generated  according  to  the  common  method  with  pure  zinc 
and  pure  sulphuric  acid,  25  to  30  per  cent.  The  precautions  advised  by  chem- 
ists for  the  generation  of  hydrogen 
must  be  carefully  followed  because 
when  hydrogen  mixed  with  oxygen 
or  air  is  ignited  a  violent  and 
disastrous  explosion  may  occur. 
Those  unfamiliar  with  the  genera- 
tion of  hydrogen  should  seek  the 
aid  of  someone  who  is  informed  so 
as  to  avoid  accidents  arising  from 
explosion.  The  hydrogen  must 
not  be  allowed  to  escape  in  the 
neighborhood  of  a  flame  for  fear 
of  explosion. 

The  well-known  Kipp's  gener- 
ator may  be  used.  First  let  the 
reservoir  fill  with  hydrogen;  then 
allow  its  contents  to  escape.  This 
should  be  repeated,  after  which 
some  of  the  hydrogen  may  be 
collected,  in  an  inverted  test-tube 
under  water.  When  this  sample  is 
ignited,  it  should  burn  without  any 
explosion;  for  if  it  explodes  this  shows  that  oxygen  is  still  mixed  with  it.  The 
hydrogen  should  bubble  through  the  medium  five  minutes  or  more. 

The  inconvenience  and  danger  of  sealing  the  tubes  in  the  flame,  as  has  to  be 
done  in  Liborius-Frankel's  and  other  methods  for  cultivation  under  hydrogen, 
are  obviated  in  Navy's  apparatus.  The  tubes  or  plates  are  placed  in  jars 
through  which  hydrogen  may  be  conducted.  The  stopper,  having  been  smeared 
previously  with  a  soft  wax,  is  sealed  by  giving  it  one-fourth  of  a  turn. 

There  have  been  various  other  kinds  of  apparatus,  usually  complicated  and 
expensive,  devised  for  the  growth  of  plate-cultures  under  hydrogen,  but  Novy's 
jars  are  the  best,  both  for  tubes  and  for  plates. 

Other  expedients  for  the  cultivation  of  anaerobic  bacteria  are  less  effective. 


FIG.  30. — Novy's  jar  for  the  cultivation 
of  anaerobes. 


THE    CULTIVATION    OF   BACTERIA.  93 

In  cases  where  a  very  deep  stab-culture  is  made  in  gelatin  or  agar,  where  the 
growth  appears  in  the  lower  part  of  the  tube  by  preference,  it  is  supposed  to  be 
anaerobic.  Koch  covered  part  of  the  surface  of  a  gelatin  plate  with  a  bit  of 
sterilized  mica  or  a  cover-glass;  bacteria  which  grew  beneath  this  place  were 
considered  to  be  anaerobic.  Another  method  was  to  cover  the  surface  of  the 
gelatin  in  the  culture-tube  with  sterilized  oil.  W.  H.  Park  has  recommended 
a  mixture  of  solid  paraffin  with  25  to  50  per  cent,  of  fluid  paraffin  or  albolene 
as  a  covering  for  the  surface  of  anaerobic  cultures.  This  mixture  has  a  semi- 


FIG.  31. — Streak  culture  of  the  potato  bacillus  (natural  size),  showing  an 
aerobic  organism  which  will  not  grow  under  a  cover-glass. 

solid  consistency,  and  does  nor  retract  at  the  edges  on  cooling.  The  paraffin 
prevents  the  absorption  of  oxygen  except  to  a  small  extent  at  the  edges.  The 
method  is  useful  for  large  quantities  of  culture  material,  as  in  flasks.  Esmarch 
advised  making  roll-tubes,  and  after  cooling  them  to  fill  them  with  a  melted 
gelatin  cooled  down  to  near  the  point  of  solidification.  Hueppe  made  use  of 
eggs  in  their  shells.  The  egg-shell  was  carefully  cleaned,  sterilized  with  a  solu- 
tion of  bichloride  of  mercury,  washed  with  sterilized  water  and  wiped  dry  with 
sterilized  cotton'.  The  end  of  the  egg-shell  was  punctured  with  a  hot  needle. 
Through  the  opening  thus  made  the  inoculation  was  accomplished.  The 
opening  was  closed  with  collodion. 


CHAPTER  V. 
CULTIVATION  OF  BACTERIA,  CONTINUED. 

Isolation  of  Bacteria.— In  order  to  study  any  kind  of 
bacteria  it  is  necessary  to  have  the  particular  species  separated 
from  other  sorts  with  which  it  may  be  mixed.  The  earlier 
bacteriologists  endeavored,  to  separate  bacteria  of  different 
sorts  of  successive  transplantations  through  a  series  of  tubes. 
The  procedure  now  generally  used  for  this  purpose  is  the  so- 
called  plate-method  of  Koch.  The  great  progress  which 
bacteriology  has  made  during  the  last  twenty  years  is  largely 
owing  to  the  use  of  this  method. 

Pathogenic  bacteria  may  sometimes  be  isolated  through 
inoculations  into  animals.  Thus  an  animal  may  be  inoculated 
with  sputum  containing  tubercle  bacilli  mixed  with  other  bac- 
teria. The  animal  may  die  of  tuberculosis,  and  its  tissues  may 
contain  tubercle  bacilli  in  pure  culture,  the  other  bacteria  hav- 
ing produced  no  important  effect. 

Still  another  method  which  is  occasionally  useful  is  to  sub- 
ject the  mixture  of  bacteria  to  a  heat  of  80°  or  90°  C.  for  a  few 
minutes.  If  it  contains  resistant  spores,  like  those  of  the  teta- 
nus bacillus  or  hay  bacillus,  they  may  be  expected  to  survive, 
and  may  be  propagated  in  pure  culture,  everything  else  having 
been  killed  by  the  heat. 

Plate-cultures. — It  is  impossible  in  most  cases  to  distin- 
guish between  bacteria  of  different  varieties  by  microscopic 
examination  alone.  Bacteria  of  widely  different  species  and 
quite  unlike  one  another  in  their  properties  may  present 
similar  appearances  under  the  microscope.  The  differences 
which  they  exhibit  are  usually  apparent  when  they  are  grown 

94 


THE    CULTIVATION   OF    BACTERIA.  95 

in  culture-media.  The  growth,  called  a  colony,  which  results 
from  the  multiplication  of  a  single  bacterium,  is  in  many  cases 
quite  characteristic  for  the  species.  By  the  plate-method  the 
individual  bacteria  in  a  mixture  are  separated  from  one  another 
by  distributing  them  through  melted  gelatin  or  agar  in  tubes. 
They  are  fixed  in  the  place  where  they  chance  to  be  when  the 
medium  solidifies.  They  are  allowed  to  grow,  and  from  each 
individual  there  forms  a  colony.  It  is  usually  possible  to  dis- 
tinguish between  colonies  arising  from  different  species  when  it 
was  not  possible  to  distinguish  between  the  individual  bacteria 
of  these  species.  A  convenient  illustration  has  been  suggested 
by  Abbott.  A  number  of  seeds  of  different  sorts  may  appear 
very  much  alike,  and  considerable  difficulty  may  be  found  in 
distinguishing  one  from  another  with  the  eye.  Let  them  be 
sown,  however,  and  let  plants  develop  from  them,  and  these 
plants  will  easily  be  distinguished  from  one  another.* 

Method  of  Making  Plate-cultures. — Melt  three  tubes  of 
gelatin  or  agar.  (There  is  some  difficulty  in  keeping  agar  in  a 
fluid  state  while  dilutions  are  being  made.  It  is  best  to  have 
some  form  of  water-bath  with  a  thermometer  for  the  purpose.) 
Let  the  melted  tubes  cool  to  a  few  degrees  above  40°  C.  Take 
a  small  portion  of  the  material  to  be  examined — pus,  for  exam- 
ple— and  introduce  it  with  a  sterilized  platinum  wire  or  loop 
into  one  of  the  tubes.  Stir  it  in  carefully.  Remove  the  needle, 
sterilize  it  and  replace  the  plug.  Mix  the  material  introduced 
thoroughly  with  the  melted  culture-medium,  taking  care  not 
to  wet  the  plug.  Now  remove  the  plug  again,  and,  having 
sterilized  the  platinum  wire,  insert  it  into  the  liquefied  medium. 
Carry  three  loopfuls  in  succession  from  this  tube,  which  is  No. 
i,  into  tube  No.  2;  sterilize  the  needle;  replace  the  plugs;  mix 
thoroughly  by  tilting  the  tube  up  and  down,  but  avoid  shaking 

*It  must  be  understood  that  no  close  comparison  can  be  drawn  between  higher 
plants,  which  simply  complete  the  development  of  parts  potentially  present  in 
the  seed,  and  colonies  o}  bacteria,  which  are  aggregates  of  individuals  the  progeny 
of  one  individual  of  the  same  kind. 


96  MANUAL    OF    BACTERIOLOGY. 

the  medium  up  into  foam,  and  also  avoid  wetting  the  plug. 
Carry  three  loopfuls  from  tube  No.  2  into  tube  No.  3  in  the 
same  manner.  The  original  material  will  obviously  be  diluted 
in  tube  No.  i,  more  in  tube  No.  2  and  still  more  in  tube  No.  3. 
The  most  convenient  form  of  plate  is  that  known  as  a  Petri  dish, 
a  small  glass  dish  about  8  cm.  in  diameter  and  1.5  cm.  in  height, 
provided  with  a  cover  which  is  a  little  larger,  but  of  the  same 
form.  This  dish  should  be  cleaned  and  sterilized  in  a  hot- 
air  sterilizer  at  150°  C.  or  higher  for  an  hour.  When  it  is  cool 
it  may  be  used. 

Such  dishes  having  previously  been  prepared,  the  contents 
of  tube  No.  i  are  poured  into  one  dish,  and  those  of  tube  No.  2 
into  another  and  those  of  tube  No.  3  into  a  third.  They  are 


FIG.  32.— Petri  dish. 

to  be  labeled  Nos.  i,  2  and  3.*  In  pouring  proceed  as  follows: 
Remove  the  plug  of  tube  No.  i ;  heat  the  neck  of  the  tube  in  the 
flame;  allow  it  to  cool,  holding  it  in  a  nearly  horizontal  position. 
When  the  tube  has  cooled,  lift  the  cover  of  the  Petri  dish  a  little, 
holding  it  over  the  dish;  pour  the  contents  of  tube  No.  i  into 
the  dish  and  replace  the  cover  of  the  dish.  The  interior  of  the 
dish  should  be  exposed  as  little  and  as  short  a  time  as  possible. 
Tubes  Nos.  2  and  3  are  to  be  treated  in  the  same  manner. 
Burn  the  plugs,  and  fill  the  empty  tubes  with  5  per  cent,  solution 
of  carbolic  acid.  They  should  be  sterilized  for  an  hour  in  the 
steam  sterilizer  on  each  of  three  days. 

*The  labels  should  be  moistened  with  the  finger,  which  has  been  dipped  in 
water.  They  should  not  be  licked  with  the  tongue.  While  working  in  the  bac- 
teriological laboratory  it  is  best  to  make  it  a  rule  that  no  object  is  to  be  put  in  the 
mouth. 


THE    CULTIVATION    OF    BACTERIA.  97 

The  culture-medium  in  the  Petri  dish  will  soon  solidify. 
Petri  dishes  of  agar  should  be  inverted  as  soon  as  the  medium 


FIG.  33. — Dilution-cultures  in  Esmarch  roll-tubes. 

In    tube   i    the   colonies  are  very  close  together;  in  tube  2  they  are  somewhat 
separate;  in  tube  3  they  are  well  isolated. 

is  thoroughly  solidified,  otherwise  the  water  which  evaporates 
7 


gg  •    MANUAL   OF    BACTERIOLOGY. 

from  the  surface  condenses  on  the  inside  of  the  lid,  and  runs 
down  over  the  surface  of  the  agar.  A  round  piece  bf  filter 
paper  placed  over  the  dish  before  putting  on  the  lid  may  also  be 
employed,  or  the  cover  may  be  made  of  porous  earthenware,  as 
recently  recommended  by  Hill.  Colonies  develop  usually  in 
from  one  to  two  days,  more  quickly,  of  course,  in  the  incubator.' 
In  plate  No.  i  they  will  be  very  numerous,  in  plate  No.  2  less 
numerous  and  in  plate  No.  3  still  less  numerous.  Where  the 
number  is  small  the  colonies  will  be  widely  separated  and  can 


FIG.  34. — Appearance  of  colonies  on  gelatin  in  Petri  dish. 

readily  be  studied.  They  may  be  examined  with  a  hand-lens, 
or  the  entire  dish  may  be  placed  on  the  stage  of  the  microscope 
and  the  colonies  be  inspected  with  the  low  power.  The  iris 
diaphragm  should  be  partly  closed  and  the  concave  mirroi 
should  be  used.  Dilution-cultures  prepared  as  described  in 
the  next  paragraph,  where  the  principle  is  the  same,  are  showr 
in  Fig.  31.  In  tube  No.  i  the  colonies  are  so  numerous  as  tc 
look  like  fine,  white  dust.  In  tubes  2  and  3  they  become  less 
numerous  and  larger. 


THE    CULTIVATION    OF    BACTERIA. 


99 


Esmarch's  Roll-tubes. — Either  gelatin  or  agar  may  be  used 
for  roll-tubes,  but  if  the  agar  is  freshly  made,  it  does  not  adhere 
well  to  the  walls  of  the  tuber.  The  dilutions  in  tubes  i,  2, 
and  3  are  made  as  above.  Tubes  containing  a  rather  small 
amount  of  the  culture-medium  are  more  convenient.  The 
tubes  may  be  capped  with  a  rubber  cap  and  revolved  in  a  dish 
of  ice  water,  or  under  the  water  tap  or,  a  still  better  method 
employed  first  by  W.  D.  Booker,  a  block  of  ice  should  be  at 
hand,  and,  with  a  tube  filled  with  hot  water  and  lying  horizon- 


FIG.  35. — Manner  of  making  Esmarch  roll-tube. 


tally,  a  hollow  of  the  size  of  the  test-tube  should  be  melted 
on  the  upper  surface  of  the  ice.  In  this  hollow  place  the  tube 
of  liquified  gelatin  or  agar;  roll  it  rapidly  with  the  hand,  taking 
care  that  the  culture-medium  does  not  run  toward  the  neck 
as  far  as  the  cotton  plug.  The  medium  is  spread  in  a  uniform 
manner  around  the  inside  of  the  tube,  where  it  becomes  solidi- 
fied. Gelatin  roll-tubes  must  be  kept  in  a  place  so  cool  that 
there  is  no  danger  of  their  melting;  in  handling  them  they 
are  to  be  held  by  the  portion  of  the  tube  into  which  the  cot- 
ton plug  projects,  so  that  the  warmth  of  the  hand  may  not 


IOO  MANUAL    OF    BACTERIOLOGY. 

melt  the  gelatin.  Agar  roll-tubes  should  be  kept  in  a  posi- 
tion a  little  inclined  from  the  horizontal,  with  the  mouth  up, 
for  twenty-four  hours,  so  that  the  agar  may  stick  to  the  wall 
of  the  tube.  For  reasons  stated  above,  it  is  best  to  employ 
agar  which  has  partially  dried  out. 

By  the  plate-method  as  originally  devised  by  Koch,  instead  of  using  Petr 
dishes,  the  gelatin  was  poured  upon  a  sterile  plate  of  glass.  This  plate  of 
glass  was  laid  on  another  larger  plate  of  gla-s,  which  formed  a  cover  for  a  dish 
of  ice-water,  the  whole  being  provided  with  a  leveling  -apparatus.  The  plate 
was  kept  perfectly  level  until  it  had  solidified,  which  took  place  rapidly  on  the 
cold  surface.  The  glass  plates  were  placed  on  little  benches  enclosed  within  a 
sterile  chamber.  This  method  is  now  seldom  or  never  used. 

The  isolation  of  bacteria  may  sometimes  be  effected  by  draw- 
ing a  platinum  wire  containing  material  to  be  examined  rapidly 
over  the  surface  of  a  Petri  dish  containing  solid  gelatin  or  agar; 
or  over  the  surface  of.  the  slanted  culture-medium  in  a  test- 
tube;  or  by  drawing  it  over  the  surface  of  the  medium  in 
one  test-tube,  then  without  sterilizing,  over  the  surface  of 
another,  perhaps  over  several  in  succession. 

Another  very  convenient  method  of  obtaining  isolated  colo- 
nies is  to  introduce  a  very  small  amount  of  material  into  the 
water  squeezed  out  in  the  bottom  of  a  slant  agar  tube,  then 
flood  this  over  the  surface  of  the  agar. 

Appearance  of  the  Colonies.— The  colonies  obtained  in 
the  Petri  dishes  or  roll-tubes  (Fig.  32)  may  be  studied  with  a 
hand-lens  or  with  a  low -power  microscope.  In  the  latter  case, 
use  the  concave  mirror  with  the  iris  diaphragm  partly  closed. 
The  colonies  present  various  appearances.  Some  of  them  are 
white,  some  colored;  some  are  quite  transparent  and  others  are 
opaque;  some  are  round,  some  are  irregular  in  outline;  some 
have  a  smooth  surface,  others  appear  granular,  and  others 
present  a  radial  striation.  Surface  colonies  often  present 
different  appearances  from  those  occurring  more  deeply.  Sur- 
face colonies  are  likely  to  be  broad,  flat  and  spreading.  If  the 


THE    CULTIVATION   OF    BACTERIA.  IOI 

colony  consists  of  bacteria  which  have  the  property  of  liquefying 
gelatin,  a  little  funnel-shaped  pit  or  depression  forms  at  the  site 
of  the  colony.  The  appearance  of  colonies  may  be  of  great  as- 
sistance in  determining  the  character  of  doubtful  species. 
The  appearance  in  gelatin  plates  of  the  colonies  of  the 
spirillum  of  Asiatic  cholera,  for  instance,  is  one  of  the  most 
characteristic  manifestations  of  this  organism. 

Pure  Culture. — From  these  colonies  pure  cultures  may  be 
obtained  by  what  is  called  "  fishing."  Select  a  colony  from 
which  cultures  are  to  be  made;  touch  it  lightly  with  the  tip 
of  a  sterilized  platinum  wire,  taking  great  care  not  to  touch 
the  medium  at'  any  other  point.  Introduce  the  wire  into  a 
tube  of  gelatin.  Sterilize  the  wire  and  plug  the  tube.  In  a 
similar  manner,  and  from  the  same  colony,  inoculate  tubes  of 
agar,  bouillon,  milk,  potato  and  blood-serum.  At  the  same 
time  it  is  well  to  make  a  smear  preparation  from  the  colony 
and  to  stain  with  one  of  the  aniline  dyes  so  as  to  determine 
the  morphology  of  the  bacteria.  The  growths  which  take 
place  in  the  tubes  should  contain  one  and  the  same  kind  of 
bacteria.  As  seen  under  the  microscope  the  bacteria  should 
have  the  same  general  form  and  appearance  as  those  seen  in 
the  colony  from  which  they  were  derived.  This  will  be  the 
case,  provided  the  colony  has  resulted  from  the  development 
of  a  single  bacterium  or  from  several  bacteria  of  the  same 
kind.  Occasionally,  however,  a  colony  will  develop  from 
several  bacteria  which  may  not  all  be  alike.  In  that  case  a 
pure  culture  will  not  be  obtained,  and  the  process  of  plating 
may  have  to  be  repeated. 


CHAPTER  VI. 
INOCULATION  OF  ANIMALS. 

IN  the  study  of  pathogenic  bacteria,  the  inoculation  of  animals 
is  frequently  indispensable.  It  is  inexpedient  where  classes 
are  large  for  students  to  make  such  inoculations;  but,  never- 
theless, every  student  should  be  familiar  with  the  subject. 
The  animals  most  often  used  are  white  mice,  guinea-pigs, 
rabbits  and  pigeons.  Larger  animals  are  also  employed  for 
special  purposes.  The  hair  in  all  cases  should  be  removed 
from  the  point  selected  for  inoculation,  and  if  the  material 
to  be  introduced  is  solid  or  semisolid.  as  with  tissue  pulp,  a 
small  V-shaped  opening  in  the  skin  is  made  with  scissors,  and 


FIG.  36. — Mouse-holder. 

a  stiff,  sterilized,  platinum  wire  or  with  the  point  of  the  scissors 
or  of  a  pair  of  forceps,  is  passed  into  this  opening,  separating 
the  skin  from  the  muscles  for  some  distance  so  as  to  make  a 
pocket.  Into  this  pocket  the  material  is  introduced  by  means 
of  the  platinum  wire.  The  wound  may  be  covered  with  collo- 
dion. Such  solid  or  semi-solid  material  may  also  be  conveni- 
ently introduced  by  placing  them  in  a  sterile  glass  cannula, 
which  is  pushed  to  the  proper  situation  through  a  small  incis- 

102 


INOCULATION    OF    ANIMALS. 


I03 


ion.     The  substance  in  the  cannula  is  forced  out  of  it  with  a 
stiff,  sterile,  platinum  wire.     (Fig.  37.) 

The  peritoneal  cavity  may  be  inoculated 
through  an  incision  in  the  abdominal  wall,  into 
which  the  desired  substance  may  be  introduced 
with  a  sterile  platinum  wire,  the  incision  being 
closed  with  sutures. 

But  a  more  convenient  method  in  many  cases, 
both  for  subcutaneous  as  well  as  intraperitoneal 
inoculations,  is  the  use  of  a  hypodermatic  syringe. 
Material  from  the  surface  of  solid  media  can  be 
suspended  in  sterile  beef -broth  or  physiological 
salt  solution,  or  tissue  pulp  may  be  mascerated 
in  the  same  liquids,  or  cultures  in  fluid  media 
used  directly  for  these  injections. 

Intravenous  inoculation  is  most  commonly 
practiced  upon  rabbits.  A  small  vein  which  is 
near  the  posterior  margin  of  the  ear  of  the  rabbit 
is  easily  reached  from  the  dorsal  surface;  the  ear 
having  been  shaved  and  washed  with  alcohol, 
the  hypodermatic  needle  is  introduced  directly 
into  this  vein.  In  making  a  hypodermatic  injec- 
tion, the  needle  and  syringe  should  of  course  be 
sterilized  before  and  after  each  operation. 

For  the  inoculation  of  mice  resort  may  be  had 
to  some  sort  of  mouse-holder  (Fig.  34),  or  the 
animal  may  be  held  by  an  assistant,  who  takes 
the  skin  at  the  back  of  the  neck  between  his 
fingers  and  at  the  same  time  holds  the  tail. 
Resort  may  be  had  to  etherization  by  using  a 
few  drops  of  the  anesthetic  on  a  bit  of  cotton 
wool  placed  in  the  jar  with  the  mouse,  but  there 
is  danger  of  killing  the  animal  in  this  way.  After 
inoculation  the  mice  may  be  kept  in  a  glass  jar  covered  with 


FIG.  37. 


104  MANUAL    OF    BACTERIOLOGY. 

wire  netting  or  *in  a  Mason  preserve  jar  with  holes  punched 
in  the  top.  They  may  be  fed  with  moistened  bread  or  oats. 
It  is  important  to  see  that  they  receive  drinking-wate^r. 

Guinea-pigs  and  rabbits  may  be  held  by  an  assistant  or  tied 
by  the  legs  upon  a  board  for  purposes  of  inoculation.  The 
hair  over  a  small  portion  of  the  abdomen  is  cut  away  and  a 
short  incision  is  made  through  the  skin;  a  pocket  is  produced 
with  a  stiff  wire,  and  the  material  inserted  with  a  sterile  plati- 
num wire.  The  wound  may  be  covered  with  collodion. 
Sutures  may  be  used  if  the  wound  is  large.  Guinea-pigs 
and  rabbits,  after  inoculation,  are  to  be  kept  in  cages  of  galva- 
nized iron  and  wire-netting.  The  bottom  may  conveniently 
be  made  in  the  form  of  a  movable  pan  which  permits  of  the 
disinfection  of  the  excreta.  Rabbits  and  guinea-pigs  may  be 
fed  with  oats,  carrots,  cabbage,  grass  and  the  like. 

Autopsies  upon  animals  should  be  held  as  soon  as  possible 
after  death.  During  the  interval  the  body  should  be  kept  in 
the  ice-box.  The  autopsy-room  should  be  furnished  with 
screens  to  keep  out  flies,  so  that  they  may  not  light  on  the  in- 
fected animal.  The  animal  should  be  extended  on  its  back 
upon  a  board.  The  legs  may  be  fastened  with  pins  or  tacks. 
The  animal  should  be  handled  with  forceps  as  far  as  possible, 
and  after  beginning  the  autopsy  the  fingers  should  not  touch  it. 
If  the  fingers  come  in  contact  with  infectious  matter,  disinfect 
them  at  once.  Have  a  basin  of  bichloride  of  mercury  solution 
i-iooo  ready  for  this  purpose.  Knives,  scissors,  platinum 
wires  and  forceps  should  be  sterilized  in  the  flame  before  and 
after  each  manipulation.  Be  prepared  to  make  smear  prep- 
arations on  cover-glasses,  and  to  inoculate  tubes  of  gelatin, 
agar  and  other  media  as  desired.  Moisten  the  hairs  over  the 
thorax  and  abdomen  with  bichloride  of  mercury  solution— 
1000,  to  prevent  them  from  being  carried  into  the  air.  Make 
an  incision,  passing  through  the  skin  from  the  sternum  to  the 
pubis  along  the  thorax  and  abdomen,  and  diagonal  incisions 


INOCULATION    OF    ANIMALS.  IO5 

extending  down  the  fore  and  hind  legs.  Dissect  away  the  skin 
from  the  thorax,  abdomen  and  upper  parts  of  the  legs.  With 
a  knife  heated  in  the  flame,  sear  a  broad  line  extending  down 
the  middle  of  the  abdomen.  Through  this  burned  surface 
make  an  incision  through  the  muscles  of  the  abdomen.  In  a 
similar  manner  make  a  transverse  incision  across  the  middle 
of  the  abdomen  through  a  burned  surface.  Inoculations 
on  culture-media  should  be  made  from  the  peritoneal  cavity, 
cover-glass  preparations  which  are  afterwards  to  be  stained 
should  be  made  by  smears  from  various  tissue  juices.  With 
a  hot  knife,  scorch  a  small  area  on  the  surface  of  the  liver; 
through  this  surface  enter  the  liver  with  a  sterilized  platinum 
wire,  and  with  the  material  thus  obtained  inoculate  the  tubes; 
also  make  cover-glass  preparations.  In  the  same  manner  inoc- 
ulate tubes  and  make  cover-glass  preparations  from  the  spleen, 
the  kidneys,  the  pleural  cavity,  the  pericardial  cavity,  the 
lungs,  and  the  blood  inside  the  heart  and  other  organs  as  indi- 
cated. If  there  is  a  question  of  the  tissues  from  which  the 
cultures  are  to  be  made  having  become  contaminated,  as  might 
be  the  case  where  the  autopsy  is  delayed  for  any  reason,  it  is 
better  to  make  plate  cultures  in  Petri  dishes  as  described  on  page 
96.  All  incisions  are  to  be  made  through  the  burned  surfaces, 
and  all  material  used  for  inoculation  is  to  be  obtained  through 
burned  surfaces.  In  sterilizing  the  instruments  in  the  flame 
avoid  sputtering,  especially  when  they  become  covered  with 
oil  from  adipose  tissue.  Pieces  of  lung,  liver,  spleen,  kidney  and 
other  organs,  as  may  be  indicated,  should  be  placed  in  95  per 
cent,  alcohol  or  10  per  cent,  formalin  for  fixation  and  hardening. 
The  animal  and  the  board  on  which  it  was  extended  should  be 
covered  with  bichloride  of  mercury  solution  i-iooo,  and 
afterward  burned.  The  cage  or  jar  and  the  instruments,  dishes 
and  towels  used  should  be  sterilized  by  steam.  The  hands  of 
the  operator  should  .be  washed  thoroughly  with  soap  and  water 
and  with  a  i-iooo  solution  of  bichloride  of  mercury,  if  there 


106  MANUAL   OF    BACTERIOLOGY. 

is  any  possibility  of  these  having  accidentally  come  in  contact 
with  any  of  the  diseased  tissues. 

Collodion  Capsules. — Bacteria  may  be  cultivated  in  the 
living  body  of  an  animal,  without  infecting  the  animal,  when 
they  are  enclosed  in  collodion  capsules.  Their  soluble  products 
are  able  to  diffuse  through  the  collodion,  while  the  animal's 


) 


FIG.  38. — Method  of  making  collodion  capsules. — (After  McCrae.} 

fluid  may  pass  into  the  sac  to  nourish  them.  These  capsules 
were  originally  made  by  dipping  the  round  end  of  a  glass  rod 
into  collodion  repeatedly.  McCrae's  method*  is  easier  and 
more  satisfactory.  (Fig.  36.) 

A  piece  of  glass  tubing  is  taken,  and  a  narrow  neck  drawn  on  it  near  one  end. 
This  end  of  the  tube  is  rounded  in  the  flame  and  while  still  warm,  the  body  of 
a  gelatin  capsule  is  fitted  over  it,  so  that  the  gelatin  may  adhere  to  the  glass.  The 
capsule  is  now  dipped  into  3  per  cent,  collodion,  covering  the  gelatin  and  part 
of  the  glass.  It  is  allowed  to  dry  a  few  minutes,  and  is  dipped  again.  In  all, 
two  or  three  coatings  may  be  given.  The  capsule  is  filled  with  water  and  boiled 
in  a  test-tube  with  water.  The  melted  gelatin  is  removed  from  the  inside  o  f  the 
capsule  by  means  of  a  fine  pipette.  The  causule  is  partly  filled  with  water  or 
broth  and  sterilised.  The  capsule  may  now  be  inoculated.  The  narrow  part 
of  the  'glass  tube  which  constitutes  the  neck  must  then  be  sealed  in  the  flame, 
taking  care  that  the  neck  be  dry.  The  sealed  capsule  should  be  placed  in  bouillon 
for  twenty-four  liours.  No  growth  should  occur  outside  the  capsule  if  it  is  tight. 
It  may  now  be  placed  in  the  peritoneum  of  an  animal. 

A  method  for  making  collodion  sacs  recommended  by  Gorslinef  consists  in 
the  use  of  a  glass  tube,  the  lower  end  of  which  is  rounded  and  closed,  except  a 
small  hole,  which  is  temporarily  filled  with  collodion.  This  tube  is  dipped  in 
collodion  and  dried,  as  above.  It  may  now  be  filled  with  water.  By  blowing  at 
the  opposite  end,  the  pressure  through  the  hole  in  the  bottom  of  the  glass  tube 
will  cause  the  capsule  to  loosen  so  that  it  comes  away  easily. 

There  are  also  various  other  methods  recommended  for  making  collodion  sacs. 


^Journal  of  Experimental  Medicine.     Vol.  VI.,  p.  635. 

fSee  Contributions  to  Medical  Research.     Dedicated  to  Victor  C.  Vaughan, 
1903. 


CHAPTER  VII. 
COLLECTION  OF  MATERIAL. 

All  material  used  for  bacteriological  examination  should 
be  fresh,  for  the  reason  that  changes  in  the  number  as  well  as 
the  kinds  of  bacteria  take  place  quickly  in  such  material  as 
furnish  a  suitable  soil  for  the  development  of  bacteria  under 
ordinary  circumstances.  Samples  of  water  or  milk  should  be 
examined  as  soon  after  drawing  as  possible;  but  when  this  is 
impossible,  as  in  the  case  where  they  are  transmitted  from  a 
distance,  they  should  be  collected  in  sterilized  tubes  or  bottles, 
which  should  be  kept  on  ice  but  not  frozen.  Specimens  of 
sputum  should  be  collected  in  clean  bottles  tightly  corked. 
The  early  morning  sputum  is  to  be  preferred  for  examination. 
The  patient  should  be  directed  to  rinse  out  the  mouth  carefully, 
and  cough  up  material  from  the  lungs,  not  merely  to  clear  out 
the  throat  as  is  sometimes  done.  It  should  be  examined  as 
soon  as  possible.  Although  decomposition  appears  not  to 
interfere  with  the  staining  properties  of  the  tubercle  bacilli, 
the  sputum  should  be  fresh  in  order  that  the  other  bacteria 
contained  in  it  may  be  studied.  Therefore,  it  should  be  free 
from  contamination  with  putrefactive  germs.  Valuable  infor- 
mation can  also  be  obtained  by  examination  of  sputum  in 
a  fresh  condition  before  staining  (see  also  page  34). 

Samples  of  urine  keep  better  after  the  addition  of  a  few 
crystals  of  thymol,  which  retards  the  fermentative  process,  so 
that  the  sedimentation  of  the  bacteria  and  of  other  solid  mat- 
ter in  conical  vessels  is  facilitated,  although  that  purpose 
can  be  accomplished  at  once  by  the  centrifuge.  Thymol  will 
also  be  a  useful  addition,  as  far  as  a  bacteriological  examina- 

107 


108  MANUAL    OF    BACTERIOLOGY. 

tion  is  concerned,  in  case  samples  of  urine  are  to  be  sent  by 
mail;  thymol  should  not  be  added  if  cultures  are  to  be  made. 
Specimens  of  sputum,  pus  or  blood  may  be  collected  con- 
veniently in  the  form  of  thin  smears  upon  cover-glasses.  The 
smears  are  fixed  by  passing  through  the  flame  three  times. 
Smears  of  blood  are  prepared  as  follows:  Have  two  perfectly 
clean,  square  cover-glasses.  The  finger,  or  the  lobe  of  the  ear, 
having  been  carefully  washed  with  water,  alcohol  and  ether,  is 
punctured  with  a  sterilized  needle,  and  a  small  drop  of  blood 
issues  which  is  wiped  away  with  a  clean  cloth.  The  second 
drop  of  blood  should  be  taken;  it  should  be  about  the  size  of  a 
pin's  head.  No  pressure  should  be  exerted  upon  the  skin. 
This  drop  of  blood  is  placed  on  one  of  the 
cover-glasses.  The  other  cover-glass  is  laid 
upon  the  first,  both  being  handled  with  for- 
ceps. The  drop  of  blood  becomes  flattened 
out  into  a  thin  film.  Immediately  and  be- 
fore the  blood  has  had  time  to  coagulate 

FiG.39.-Mannerof    the  two  are  sliPP^d  or  slid  away  from  each 
placing   cover-glasses    other   in    a   horizontal  plane,  not  forciblv 

in    making    films    of  ,  T  J 

blood.— (After  Cabot.)  pulled  apart.  In  this  way  the  blood  will  be 
spread  in  thin  films  on  the  cover-glasses. 
It  is  best  to  place  the  cover-glasses  so  that  one  does  not  cover 
the  other  exactly,  but  so  that  the  sides  of  the  one  lie  diagonally 
to  the  sides  of  the  other,  although  their  centers  coincide  (Fig. 
37).  Films  of  blood  which  are  to  be  examined  for  the  parasite 
of  malaria  may  be  prepared  in  this  manner.  Drops  of  blood 
to  be  used  for  the  serum  reaction  for  typhoid  fever  must  be 
large-sized.  They  may  be  collected  on  cover-glasses  or  pieces 
of  unsized  paper  and  allowed  to  dry.  In  place  of  drops  of 
blood  caught  in  this  manner,  the  use  of  capillary  tubes  for 
collecting  the  blood  are  to  be  preferred,  since  by  this  method 
it  is  possible  to  obtain  the  serum  free  from  corpuscles  after  the 
clot  forms  in  the  tube.  To  test  blood  by  culture  methods 


COLLECTION  OF  MATERIAL.  109 

not  less  than  10  c.c.  of  blood  should  be  taken.*  This  is  most 
conveniently  accomplished  by  using  a  large  hypodermatic 
needle,  and  aspirating  the  blood  from  a  vein  at  the  bend  of  the 
elbow — under  strict  antiseptic  precautions.  A  bandage  tied 
tightly  around  the  arm  above  the  elbow  facilitates  the  operation. 
The  blood  thus  taken  may  then  be  used  for  cultures  in  various 
ways.  A  good  method  for  general  purposes  is  to  empty 
the  syringe  quickly  into  a  flask  holding  100  c.c.  or  more  of  bouil- 
lon or  dextrose-bouillon.  The  mixture  of  blood  and  bouillon 
should  be  placed  in  the  incubator  for  one  to  two  days.  If  the 
the  bacteria  develop,  they  may  be  secured  in  pure  cultures  by 
plating,  and  may  be  studied  further,  as  the  occasion  requires. 

At  autopsies  on  human  subjects  the  same  principles  apply 
as  in  the  case  of  autopsies  upon  animals  (see  pages  104  and  109). 
Plate-cultures  should   be   made,  if 
possible,  directly  from  the  organs. 
In  all  cases  organs  should  be  seared 
with  a  hot  spatula  over  the  point 
where  the  platinum  wire  is  inserted.        FIG.  40.— Sternberg  bulb. 
The  method  of  isolation  by  streak- 
ing the  platinum  wire  containing  the  material  under  examina- 
tion lightly,  several,  times,  over  the  surface  of  an  agar  plate, 
will  be  found  convenient.     A  still  more  convenient  method 
is  to  inoculate  the  water  which  is  collected  at  the  bottom  of  a 
stout  agar  tube  and  flood  it  over  the  surface  of  the  agar.     In 
the   tubes,  inoculated  in  this  isolated  colonies  grow  out  on  the 
surface  of  the  slanted  agar.     At  the  same  time  smears  should 
be  made  from  the  organs  upon  cover-glasses  for  microscopic 
study,    and    portions    of    the    organs    should    be   saved  and 
hardened  in  alcohol  or  formalin. 

A  convenient  device  for  the  collection  of  infected  material  is 
a  stiff  wire  wound  with  a  pledget  of  absorbent  cotton  at  one  end, 

*For  examination  of  blood  for  typhoid  bacilli  see  Libman.     Bui.   Johns 
Hopkins  Hosp.  Vol.  XVII.,  July,  1906. 


110  MANUAL    OF    BACTERIOLOGY. 

the  whole  sterilized  in  a  tube,  as  recommended  by  Warren 
for  collecting  pus  and  other  fluids  for  examination,  and  as  in- 
troduced by  W.  H.  Park  for  the  collection  of  material  from 
the  throat  in  cases  of  suspected  diphtheria  (Fig.  83). 

The  so-called  Sternberg  bulb*  is  valuable  for  the  collection 
of  fluid  materials  for  examination.  A  piece  of  glass  tubing 
is  taken  and  drawn  out  to  a  long,  fine  tube,  and  a  bulb  blown 
at  the  other  end.  To  introduce  the  substance  into  the  bulb, 
the  expanded  end  is  heated  in  the  flame;  the  point  introduced 
below  the  surface  of  the  fluid  which  is  to  be  collected;  as  the 
bulb  cools,  the  air  in  it  contracts  and  draws  the  fluid  into  it. 
When  it  has  taken  up  as  much  as  it  will,  the  point  may  be  sealed 
off  in  the  flame.  If  it  is  to  sent  to  a  distance  and  the  same 
precautions  should  be  used  by  those  performing  the  autopsy 
to  guard  against  becoming  infected.  The  hands  should  be 
protected  with  rubber  gloves.  It  should  be  so  packed  that 
breakage  or  leakage  is  impossible,  particularly  when  infectious 
material  is  to  be  transported. 

Concerning  the  transmission  of  materials  containing  bacteria 
in  the  mails,  the  ruling  of  the  post-office  department  of  the 
United  States,  March  2,  1900,  is  a  follows: 

"That  the  order  of  the  Postmaster  General  of  December  27,  1897  (Order 
No.  677),  amendinng  Order  No.  88  of  February  5,  1896,  prescribing  the  condi- 
tions under  which  specimens  of  "diseased  tissues  may  be  admitted  to  the  mails 
is  hereby  further  modified  in  the  following  manner: 

"Specimens  of  diseased  tissues  may  be  admitted  to  the  mail  for  transmission 
to  United  States,  State,  or  municipal  laboratories,  only  when  enclosed  in  mailing 
packages  constructed  in  accordance  with  the  specifications  hereinafter  enu- 
merated: Liquid  cultures,  or  cultures  of  microorganisms  in  media  that  are 
fluid  at  the  ordinary  temperature  (below  45°  C.  or  1 13°  F.)  are  unmailable.  Such 
specimens  may  be  sent  in  media  that  remain  solid  at  ordinary  temperatures. 

"Upon  the  outside  of  every  package  shall  be  written  or  printed  the  words 
'Specimen  for  Bacteriological  Examination.  This  package  to  be  treated  as 
letter  mail.'  No  package  containing  diseased  tissue  shall  be  delivered  to  any 
representative  of  any  of  said  laboratories  until  a  permit  shall  have  first  been  issued 

*These  bulbs  were  first  recommended  by  Fliigge.  Die  Mikrooganismen. 
i  Auflage,  p.  662.  1886. 


COLLECTION    OF    MATERIAL. 


Ill 


by  the  Postmaster  General  certifying  that  said  institution  has  been  found  to  be 
entitled,  in  accordance  with  the  requirements  of  this  regulation,  to  receive  such 
specimens." 

The  regulation  includes  not  only  cultures,  but  "specimens 
of  diseased  tissues."  The  specifications  prescribing  the  man- 
ner of  packing,  which  are  minute  and  complicated,  may  be 
obtained  from  local  postmasters. 


CHAPTER  VIII. 
SYSTEMATIC  STUDY  OF  SPECIES  OF  BACTERIA.* 

IN  order  to  conduct  the  study  of  any  species  of  bacteria  it  is 
necessary  to  have  the  organism  isolated  in  a  pure  culture. 
This  is  best  accomplished  by  the  plate  method  already  de- 
scribed. Having  thus  obtained  the  organism  in  pure  culture, 
it  is  to  be  examined  with  reference  to  its  behavior  in  certain 
particulars.  It  is  well  for  the  beginner  to  study  a  few  known 
species  of  saprophytes  obtained  from  some  reliable  laboratory 
in  pure  culture.  The  points  which  are  to  be  considered  can 
be  illustrated  best  by  presenting  them  in  tabular  form,  filling 
out  the  items  of  the  table  for  a  given  species  of  bacteria. 

1.  Name. 

2.  Habitat  or  source. 

3.  Morphology;   grouping,    as   in   chains   or  in   zooglceae. 

4.  Size. 

5.  Staining    properties.     Behavior    by    Gram's    method. 

6.  Capsule,   present  or  otherwise. 

7.  Spore  formation. 

8.  Motility,  flagella. 

Observations  as  to  morphology,  grouping,  size,  staining  prop- 
erties, and  motility  should  all  be  made  on  fresh  cultures.  Agar 
cultures  from  18  to  24.  hours  old  are  usually  selected  for  the 
purpose.  This  rule  cannot  always  be  adhered  to  from  the 
nature  of  the  case  as,  for  example,  in  the  examination  of  cultures 
of  the  tubercle  bacillus,  of  the  gonococcus,  of  the  diphteria  bacil- 
lus. Examination  for  the  presence  of  spores  may  also  require 
older  cultures. 

*See  Appendix  I.,  for  recommendations  of  Society  of  American  Bacteriologists. 

112 


SYSTEMATIC    STUDY    OF    SPECIES    OF    BACTERIA.  113 

Growth  on  culture-media. 
9.  Relation    of    growth    to    temperature. 
10.  Gelatin;  observe  whether  the  gelatin  is  liquefied  or  not. 
Colonies  in  gelatin  plains,  study  under  low  power  of 
microscope. 

n.  Agar.     Colonies  in  agar  plates,  study  under  low  power 
of  microscope. 

12.  Bouillon;  note  cloudiness,  pellicle  or  precipitate. 

13.  Milk;  observe  the  reaction  and  whether  or  not  the  milk  is 

coagulated  and  subsequently  peptonized. 

14.  Production  of  gas  in  fermentation-tube  with  bouillon 

containing  sugar,  as  dextrose,  or  in  agar  with  sugars. 

15.  Potato. 

1 6.  Blood-serum;   observe   whether   or   not   peptonization 

occurs. 

17.  Production  of  indol. 

1 8.  Pigment  formation. 

19.  Production  of  acid  or  alkali. 

20.  Relation  to  oxygen;  observe  whether  the  superficial  or 

the  deep  part  of  the  growth  is  the  more  luxuriant  in 
stab-cultures;  use  anaerobic  methods  if  necessary. 

21.  Pathogenesis. 

In  commencing  the  study  of  bacteriology  the  pupil  should  try 
the  common  staining  methods  and  make  the  most  important 
culture-media.  Having  culture-media  prepared,  it  is  custom- 
ary to  study  a  number  of  species  of  non-pathogenic  bacteria. 
Notes  of  the  work  and  sketches  showing  the  morphology  of  the 
organisms  should  be  made.  In  this  as  in  other  work  with  the 
microscope,  the  value  of  even  crude  drawings  is  very  great  as  a 
matter  of  training.  It  is  well  to  choose  species  which  have 
properties  decidedly  different  from  one  another.  The  micro- 
cocci,  bacilli  and  spirilla  should  be  represented;  forms  that  are 
motile  and  that  are  not;  species  that  form  spores  and  others 
that  do  not  form  spores;  some  that  liquefy  gelatin  and  some 


114  MANUAL    OF    BACTERIOLOGY. 

that  do  not.  There  should  be  chromogenic  forms  ,  and  species 
that  ferment  dextrose,  and  that  produce  indol, — such  species 
as  some  of  the  sarcinae,  the  bacillus  coli  communis,  the  hay 
bacillus,  the  potato  bacillus,  bacillus  prodigiosus,  a  bacillus 
fluorescens  and  spirillum  rubrum.  It  is  well,  when  possible, 
to  obtain  material  directly  from  nature  rather  than  from 
laboratory  cultures:  This  may  readily  be  done  in  the  case  of 
the  hay  bacillus  and  the  potato  bacillus.  Fecal  matter  may  be 
spread  on  gelatin  plates  and  the  bacillus  coli  communis  ob- 
tained in  pure  culture.  Fluorescing  bacilli  are  very  common 
in  water.  Large  spirilla  are  often  found  in  swamp  water. 
Some  organisms  like  spirillum  rubrum  can  only  be  had  from 
laboratory  cultures.  An  instructive  experiment  which  any- 
one may  carry  out  is  to  boil  a  potato  thoroughly,  and  cut  it 
into  slices,  placing  these  on  moist  filter-paper  on  glass  plates, 
or  on  saucers,  and  after  exposing  them  to  the  air  for  half  an 
hour  or  more  to  cover  them  each  with  an  inverted  tumbler. 
Some  of  the  slices  prepared  in  this  way  should  be  put  in  the 
incubator,  others  left  at  room  temperature.  In  a  shorter  or 
longer  time  there  usually  develops  a  great  variety  of  isolated 
colonies  from  the  bacteria  that  have  fallen  on  the  slices  of  potato. 
The  growth  of  some  aerobic  organism,  like  the  potato  bacillus, 
may  be  tested  under  a  cover-glass  (see  Fig.  29).  The  pyogenic 
bacteria,. which  can  easily  be  isolated  from  pus,  may  be  studied 
in  this  connection  with  great  advantage.  The  staphylococcus 
pyogenes  aureus  and  the  streptococcus  pyogenes  should  on  no 
account  be  omitted.  The  diplococcus  of  pneumonia  can  most 
readily  be  obtained  from  a  mouse  or  a  rabbit  which  has  died 
with  pneumococcus  infection.  Such  an  animal  can  best  be 
infected  by  subcutaneous  inoculation,  using  some  of  the  rusty 
sputum  of  a  case  of  lobar  pneumonia.  The  cultivation  of  the 
pneumococcus  will  be  found  to  present  difficulties  in  classes 
containing  large  numbers  of  students. 

Representative  forms  of  moulds  and  yeasts  should  be  studied 


SYSTEMATIC    STUDY    OF    SPECIES    OF    BACTERIA.  115 

at  the  same  time.  Moulds  are  easily  obtained  by  exposing 
some  nutrient  substance  to  the  air,  covering  it,  and  allowing 
cultures  to  develop;  yeasts  will  probably  grow  also.  Ordinary 
brewer's  yeast  may  be  isolated  in  pure  culture  from  gelatin 
plates.  Bacteriological  examinations  also  should  be  made  of 
air,  soil,  water  and  milk.  With  such  simple  means,  many  of 
the  important  properties  of  bacteria  may  be  demonstrated.  It 
is  most  important  that  medical  students  should  convince  them- 
selves by  experiment  of  the  extent  to  which  bacteria  are  dis- 
seminated in  our  environments.  The  bearings  of  such  observa- 
tions on  the  practice  of  surgery  and  hygiene  are  obvious. 

Experiments  in  sterilization  and  disinfection  as  described  in 
Chapter  VIII.,  Part  II.,  may  be  performed  with  the  bacteria 

j  mentioned,  which  present  every  variety  of  resisting  power  up  to 
the  almost  incredible  resistance  of  the  spores  of  the  hay  and 

••  potato  bacilli.  The  efficiency  of  the  methods  used  for  steriliz- 
ing surgical  materials,  as  silk  and  catgut  (Chapter  IX.,  Part  II.), 

j  should  be  tested;  also,  of  the  methods  for  disinfectingthe  hands, 

if  possible,  of  the  methods  for  disinfecting  rooms,  as  well. 

After  some  proficiency  has  been  acquired,  various  pathogenic 

i  bacteria  may  be  studied  as  the  circumstances  of  the  case  re- 

|  quire.  Much  judgment  has  to  be  used  in  allowing  students 
to  work  with  pathogenic  bacteria.  Anthrax,  glanders,  tetanus, 
cholera,  bubonic  plague,  Malta  fever,  and  diphtheria  all  have 
occurred  in  laboratory  workers  through  accidental  infection, 
sometimes  with  fatal  results.  Everything  should  be  handled 
with  forceps  as  far  as  possible,  and  the  forceps  sterilized  in  the 
flame  before  and  after  using.  Particles  of  cotton  fiber  should 
not  be  allowed  to  fly  off  from  the  plugs.  The  various  rules 
for  the  management  of  the  platinum-wire,  hanging-drop  slides 
and  sputum  bottles,  and  for  the  handling  of  cultures  and  other 
infectious  materials  have  already  been  given  (pages  22,  23, 
34  and  78  to  82).  As  the  risks  of  infection  from  neglect  of 
proper  caution  are  obvious  enough,  it  would  seem,  that  it 


Il6  MANUAL   OF    BACTERIOLOGY. 

should  be  superfluous  to  warn  students  of  the  danger  to  them- 
selves and  others  of  infecting  their  hands  and  surroundings; 
but  some  who  work  in  bacteriological  laborities  become  careless, 
just  as  do  those  who  work  with  explosives.  The  most  impor- 
tant precaution,  perhaps,  is  observance  of  the  rule  that  while 
working  in  the  laboratory,  nothing  should  be  put  in  the  mouth. 
Cultures  should  never  be  left  in  improper  places.  Cultures 
of  bacteria  should  be  thoroughly  sterilized  before  the  tubes  are 
cleaned.  In  some  laboratories  tubes,  dishes  and  other  appara- 
tus, after  use,  are  placed  in  the  autoclave  or  in  the  dry  sterilizer 
or  they  are  soaked  in  disinfecting  solutions;  there  seems  to  be 
no  uniform  practice  in  this  respect. 

In  taking  these  measures,  the  same  kind  of  reasoning  applies 
as  that  which  induces  engineers  to  give  bridges  several  times 
the  strength  they  need  to  bear  the  greatest  strain  likely  to  be 
put  upon  them,  or  to  make  the  boiler  of  a  steam  engine  strong 
enough  to  bear  six  times  the  greatest  pressure  which  it  is  ex- 
pected that  the  steam  contained  in  it  will  exert. 


PART  II. 


CHAPTER  I. 

CLASSIFICATION ;  GENERAL  MORPHOLOGY 
AND  PHYSIOLOGY  OF  BACTERIA. 

IT  has  been  universally  agreed  to  class  the  bacteria  as  plants 
although  they  show  resemblances  to  both  plants  and  animals. 
On  the  one  hand,  they  seem  allied  to  the  algae  and  fungi, 
and,  on  the  other,  to  the  protozoa. 

The  classification  of  the  larger  animals  and  plants  is  based 
chiefly  upon  their  morphology.  With  the  bacteria  this  method 
of  classification  is  applicable  only  to  a  limited  extent  owing  to 
their  extreme  minuteness.  And  in  addition  it  is  necessary  to 
resort  to  the  grouping  of  the  individuals  as  seen  under  the  micro- 
scope. The  difficulty  in  classification  extends  also  to  the  mat- 
ter of  distinguishing  one  species  of  bacterium  from  another. 
Where  no  constant  difference  in  morphology  exists,  resort  is 
made  to  the  grouping  of  the  individuals  as  seen  under  the  micro- 
scope, the  presence  or  absence  of  independent  motion,  the 
naked-eye  appearance  of  the  growth  upon  culture-media  and 
their  physiological  properties  in  relation  to  growth  under 
various  conditions  of  temperature,  nutrition  and  relation  to 
free  oxygen.  The  agglutination  of  a  species  of  bacterium 
by  blood-serum  specific  for  the  speeies  (see  Chapter  VII., 
Part  II.)  has  also  been  used  for  purposes  of  identification. 

These  means  of  differentiation  are  not  entirely  satisfactory, 
and  it  is  likely  that  forms  which  are  now  considered  as  different 
species  are  not  really  such  in  all  cases.  Notwithstanding 
the  unsatisfactory  condition  of  the  classification  of  bacteria, 

117 


Il8  MANUAL   OF    BACTERIOLOGY. 

it  must  not  be  supposed  that  the  species  of  bacteria  are  not 
permanent.  For  instance,  it  would  be  incorrect  to  imagine 
that  the  micrococci  and  spirilla  become  converted  into  species 
of  bacilli,  or  for  the  bacilli  of  one  species  to  be  transmuted 
into  those  of  another.  This  is  not  in  conflict  with  the  statement 
that  we  may  frequently,  through  erroneous  and  imperfect  in- 
formation, be  in  the  habit  of  including  unlike  species  under  one 
name,  or  of  classifying  mere  varieties  of  one  species  as  entirely 
different  species.  At  present  it  is  sufficient  for  practical  pur- 
poses to  divide  bacteria  into  two  great  groups  —  the  lower  bacteria 
and  the  higher  bacteria;  and  to  subdivide  the  lower  bacteria 
into:  micrococci,  spherical  forms;  bacilli,  rod-shaped  forms, 


• 

Staphylococci.     Streptococci.     Diplococci.     Tetrads.        Sarcinae. 

FIG.  41. 

one  diameter  being  in  excess  of  the  others;  spirilla,  twisted 
like  a  corkscrew,  making  long  spirals  or  simply  parts  of  spirals 
(comma-shaped  forms).* 

Recent  investigations  indicate  that  several  species  of  bacteria 
often  are  closely  related  to  one  another,  so  as  to  form  a  well- 
marked  group.  Such  a  group  is  constituted  by  the  bacillus 
of  typhoid  fever,  bacillus  coli  communis  and  similar  forms. 
The  spirillum  of  cholera  and  other  comma-shaped  spirilla 
resembling  it  may  be  held  to  constitute  another  group.  Still 
another  is  that  containing  the  tubercle  bacillus,  smegma  bacillus 
and  other  acid-proof  bacilli. 

The  micrococci  are  subdivided  into  staphylococci,  where 
the  spheres  grow  in  clusters  like  a  bunch  of  grapes;  strepto- 
cocci, where  they  are  arranged  in  long  rows  or  chains,  like  a 

*Migula's  system  of  classifying  bacteria  has  found  favor  with  many  writers. 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.  119 

string  of  beads;  diplococci,  or  pairs  of  micrococci;  tetrads, 
where  the  individual  spheres  are  grouped  in  fours;  sarcina, 
where  they  are  grouped  in  eights  making  the  outline  of  a  cube, 
resembling  a  bale  or  package  tied  with  rope. 

The  bacilli  are  not  subdivided  in  this  manner,  although 
their  forms  vary  considerably.  The  ends  are  sometimes 
square,  sometimes  round.  Sometimes  they  are  very  short. 


&  ^ 


FIG.  32. — Bacilli  of  various  forms. 

Sometimes  they  grow  in  longer,  threadlike  forms,  in  which, 
however,  the  transverse  markings  which  indicate  the  outlines  of 
the  individual  bacilli  can  generally  be  seen,  and  which  re- 
semble a  bamboo  rod.  Short,  oval  bacilli  may  look  exceed- 
ingly like  micrococci.  Bacilli  with  rounded  extremities,  placed 
end  to  end,  look  like  strings  of  sausauges.  Under  exceptional 
circumstances,  branching  forms  of  the  bacilli  of  diphtheria, 


V 


FIG.  43.— Spirilla  of  various  forms. 

tuberculosis,  glanders  and  bubonic  plague  and  various  other 
species  have  been  encountered.* 

The  word  "bacterium"  was  formerly  used  to  designate  short 
bacilli  which  generally  formed  no  spores,  while  the  word  "bacil- 
lus" was  restricted  to  the  longer  forms  in  which  spore  formation 

*See  Hill.  Journal  o)  Medical  Research.  Vol.  VII.,  January,  1902.  Loeb. 
Ibid.  Vol.  VIII.  1902. 


120  MANUAL    OF    BACTERIOLOGY. 

occurred.  This  use  is  no  longer  common,  although  not  rarely 
the  name  bacterium  is  still  given  to  a  species — for  instance, 
bacterium  coli  commune. 

Spirilla  present  a  very  great  variety  of  form.  The  short 
" comma- shaped  bacilli"  are  only  parts,  at  most,  of  spirals,  al- 
though the  microbes  of  cholera  do  sometimes  form  long  spirals. 
On  the  other  hand,  there  are  among  spirilla  large  and  long 
sinuous  figures  which  present  most  remarkable  pictures  under 
the  microscope;  for  example,  the  spirillum  of  relapsing  fever. 
Spirilla  without  very  marked  windings  are  sometimes  called 


FIG.  44. — Involution  forms  of  the  spirillum  of  cholera. — (Van  Ermengem.} 

"vibrios";  and  long,  wavy  forms  with  corkscrew-like  windings 
"spiroch&ta"j'  and  only  the  rigidly  spinal  forms  "spirilla." 

Besides  the  purely  morphological  classification  already 
mentioned,  bacteria  are  sometimes  grouped  according  to  cer- 
tain physiological  qualities.  In  general  botany,  saprophytes 
are  plants  that  grow  on  decaying  vegetable  matter.  In  a 
bacteriological  sense,  saprophytes  are  bacteria  which  grow  in 
external  nature  on  dead  organic  matter,  and  parasites  are 
bacteria  which  exist  upon  the  living  tissues  or  fluids  of  any 
organism.  Nearly  synonymous  with  the  above  words  are  those  | 
which  do  not  and  those  which  do  produce  disease,  or  non- 
pathogenic  and  pathogenic.  The  adjectives  facultative,  or 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.  121 

optional,  and  obligate,  or  strict,  are  used  to  qualify  the  above 
terms  and  many  others. 

Size. — Bacteria  vary  greatly  in  size.  The  micrococci  are 
usually  i  p  or  less  in  diameter.  The  short  diameters  of  bacilli 
and  spirilla  also  are  less  than  i  n,  as  a  rule,  while  the  length  may 
be  several  micra.  The  anthrax  bacillus  (1.5  /*  X  3  to  10  yu)  and 
the  spirillum  of  relapsing  fever  are  the  largest  bacteria  known 
to  be  pathogenic  to  man.  To  say  that  a  microccus  is  i  n  in 
diameter  means  that  25,000  end  to  end  would  make  a  line  i 
inch  long.  It  has  been  estimated  that  i  milligram  of  a  pure 
culture  of  the  staphylococcus  pyogenes  aureus  contains 
8,000,000,000  micrococci. 

There  is  good  reason  for  believing  that  organisms  exist  which 
are  too  small  to  be  visible  with  the  most  powerful  microscopes. 
The  nature  of  these  organisms  is  not  known,  but  it  is  not 
improbable  that  some  of  them  are  bacteria.  (See  pleuro- 
pneumonia  of  cattle  etc.,  Part  II.,  Chapter  V.) 

In  stained  preparations  the  bodies  of  bacteria  frequently 
seem  to  be  homogeneous.  On  the  other  hand,  they  may  ex- 
hibit certain  spots  which  stain  more  intensely  than  others,  the 
stained  spots  alternating  with  clear  areas.  The  dark-staining 
granules  may  take  a  slightly  different  shade  of  color  from  the 
rest  (metachromatic  granules,  Babes-Ernst  bodies).  Some- 
what similar  appearances  may  result  from  changes  in  the 
density  of  the  protoplasm  of  bacteria,  leaving  vacuoles  that  do 
not  stain  (plasmolysis). 

In  old  cultures  bacteria  are  likely  to  show  irregular  and  often 
bizarre  shapes,  and  these  are  called  involution  forms.  It  is-  not 
uncommon  for  bacteria  to  be  enclosed  in  a  kind  of  envelope  of 
some  clear  substance,  which  stains  with  difficulty  or  not  at  all, 
called  a  capsule.  The  paired  micrococci  of  pneumonia  are 
enclosed  in  such  capsules.  The  capsule  is  more  likely  to  be 
demonstrated  when  the  bacteria  are  obtained  from  the  fluids 
derived  from  an  animal's  body  than  when  they  have  been 


122  MANUAL   OF    BACTERIOLOGY. 

grown  artificially  in  culture-media.  A  zooglcea  is  a  large  mass 
of  bacteria  in  a  resting  condition  held  together  by  a  mucilagi- 
nous substance.  The  composition  of  bacteria  varies  con- 
siderably with  different  species.  The  basis  appears  to  be 
proteid  substance. 

Vegetative  Cells. — All  the  forms  enumerated  above  are 
called  vegetative  cells  in  contradistinction  to  spores  to  be  de- 
scribed later,  and  multiplication  takes  place  by  the  direct 
division  or  fission  of  these  cells.  In  the  rod-shaped  bacteria 
the  fission  is  transverse.  The  formation  of  tetrads  or  sarcime 
from  micrococci  depends  upon  fission  in  two  or  three  planes. 
Repeated  fissions  of  microccocci  in  one  plane  result  in  the  for- 
mation of  streptococci.  Micrococci 
that  have  recently  divided  are  likely  to 
I  n  ^e  somewnat  flattened  on  their  op- 
posing  surfaces.  Multiplication  under 
C""")  favorable  circumstances  may  take  place 
at  an  exceedingly  rapid  rate.  Bacilli 

FIG.   45. — Bacteria    with      ,  ,  ,  ,  ,.    .  , 

capsules.  have  been  observed  to  divide  in  twenty 

minutes.    If  division  takes  place  once  in 

an  hour,  the  progeny  of  one  organism  at  the  end  of  twenty-four 
hours  will  be  16,777,216,  i.  e.,  (2  X  i  )24.  The  ordinary  form 
of  reproduction  by  fission  is  called  vegetative,  and  bacteria  that 
are  multiplying  in  this  manner  are  often  spoken  of  as  being  in 
the  vegetative  condition. 

Spores. — Under  certain  circumstances  the  reproduction  of 
bacteria  takes  place  by  means  of  the  germination  of  bodies 
called  spores.  These  appear  in  a  typical  form  in  the  large 
bacilli,  where,  near  the  centers  of  the  bacilli,  highly  refracting, 
shining  spots  may  be  seen  which  are  found  to  stain  less  readily 
with  the  aniline  dyes  than  the  rest  of  the  bacilli.  They  are  not 
to  be  confused  with  the  unstained  spots  described  as  vacuoles. 
On  account  of  their  being  formed  from  a  part  of  the  interior 
of  the  bacterium,  such  spores  are  called  endogenous.  These 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.  123 

spores  are  found  mostly  in  the  bacilli,  rarely  in  spirilla.  They 
are  what  is  meant  when  the  word  spore  is  used  alone  without 
qualification.  The  existence  of  another  kind  of  spore,  de- 
scribed as  forming  from  the  whole  of  the  bacterium  (called 
arthrospore) ,  is  doubtful.  At  all  events,  its  significance  is  not 
at  present  understood.  Spores  develop  generally,  though  not 
always,  under  adverse  conditions  of  various  kinds,  as  of  tem- 
perature and  of  nutrition.  They  are  more  resistant  to  un- 
favorable influences  of  all  sorts  than  are  the  fully  developed 
bacteria.  Spores,  as  a  rule,  resist  drying,  light,  heat  and 
chemical  agents  .to  a  remarkable  degree,  but  the  spores  from 
one  and  the  same  organism  often  differ  in  resisting  power  in 


FIG.  46. — -Bacteria  with  spores. 

different  cultures.  Frankland*  found  that  anthrax  spores 
which  formed  at  18-20°  C.  much  more  resistant  than  anthrax 
spores  formed  at  35-38°  C.  Even  individual  spores  in  the 
same  culture  differ  in  resisting  power. 

Anthrax  spores  are  said  to  have  been  found  which  could 
withstand  steam  for  twelve  minutes,  i-iooo  mercuric  chloride 
for  nearly  three  days,  or  5  per  cent,  carbolic  acid  for  more  than 
forty  days.f  The  greatest  resistance  is  displayed  by  the  spores 
of  some  of  the  saprophytic  bacteria,  particularly  those  of  hay 
and  potato,  which  are  sometimes  not  destroyed  by  several 
hours  of  steaming;  and  bacteria  which  resisted  100°  C.  for  six- 
teen hours  are  said  to  bave  been  obtained  from  the  soil.  When 

*Frankland.     Centralblat  fr  Bakteriologie,etc.     Bd.  XV.,  1894,  p.   no. 
tGiinther.     Einfiihrung  in  das  Studium  der  Bakteriologie.     Leipzig,  1906, 
p.  406. 


124  MANUAL   OF   BACTERIOLOGY. 

cultivated  at  a  temperature  as  high  as  42°  C.  for  a  sufficiently 
long  time,  the  anthrax  bacillus  becomes  incapable  of  forming 
spores;  but  as  long  as  it  retains  any  virulence  at  all  it  remains 
capable  of  forming  spores.*  Spores  themselves  do  not  mul- 
tiply, nor  do  they  manifest  any  activity.  They  may  be  located 
at  the  center  of  the  bacillus  or  nearly  at  one  end;  in  the  latter 
case  the  end  of  the  bacillus  is  likely  to  enlarge,  making  a  form 
having  the  shape  of  a  drumstick;  this  is  seen  in  tetanus  bacilli 
(Fig.  44).  When  a  bacillus  assumes  a  spindle  shape  on  ac- 
count of  having  the  middle  part  bulged  through  the  formation 
of  a  spore  it  is  called  a  dostridium.  With  rare  exceptions,  a 
single  bacillus  contains  but  one  spore.  Under  favorable  con- 


^ 

FIG.  47. — Bacteria  showing  flagella 


ditions  the  spores  germinate,  as  it  is  called,  and  develop  to  the 
adult  form  of  the  organism.  This  may  be  witnessed  in  hang- 
ing-drop preparations. 

Spore  formation  is  not  a  method  of  multiplication,  since 
one  spore  when  it  germinates  reproduces  but  one  cell,  and 
this  cell  then  multiplies.  So  spore  formation  seems  to  be  a 
means  of  preserving  the  organism  under  unfavorable  environ- 
ments, and  is  not  a  process  of  reproduction  in-  a  strict  sense. 

Motility. — Motility  is  rarely  exhibited  by  micrococci;  some 
bacilli  possess  it  and  some  do  not;  while  nearly  all  of  the 
spirilla  are  motile.  The  phenomenon  is  observed  in  the  hang- 
ing-drop. The  degree  of  motility  is  variable,  being  sometimes 
slight  and  sometimes  very  active.  When  seen  under  a  high 
power  the  little  particles  taken  from  a  culture  of  a  motile 

*Kolle  and  Wassermann.     loc.  cit.    p.  42. 


MORPHOLOGY    AND    PHYSIOLOGY   OF    BACTERIA.  125 

organism  may  look  like  a  writhing  mass  of  maggots  or  like 
tadpoles  in  a  pool..  The  motility  is  most  active  in  young 
cultures.  The  movement  results  from  the  vibration  of  little 
processes,  or  flagella  (Fig.  45) .  Of  these  there  may  be  one  or 
several,  placed  singly  or  in  groups,  at  the  end,  or  scattered 
around  the  sides.  They  are  extremely  difficult  to  demonstrate 
except  by  special  staining  methods,  which,  furthermore,  are 
quite  uncertain  of  result.  After  the  flagella  have  been  stained, 
the  bacteria  appear  somewhat  larger  than  when  stained  by  the 
ordinary  methods.  The  flagella  upon  the  bacilli  of  typhoid 
fever  are  numerous  and  form  a  very  striking  picture. 

Chemotaxis. — Motile  bacteria  possess  the  property  of  being" 
attracted  by  certain  substances  (positive  chemotaxis)  and  of 
being  repelled  by  others  (negative  chemotaxis) .     Similar  prop- 
erties are  widely  distributed  among  living  cells,  both  animal 
and  vegetable. 

FAVORABLE  AND  UNFAVORABLE  CONDITIONS  FOR  GROWTH. 

Warmth. — Among  the .  different  kinds  of  bacteria  forms 
exist  which  multiply  at  temperatures  as  low  as  o°  C.,  while 
there  are  species  that  multiply  at  70°  C.  Bacteria  which 
flourish  at  a  very  high  temperature  (maximum  about  70°  C.) 
are  called  thermophilic.  The  pathogenic  bacteria  usually 
flourish  better  at  a  point  somewhere  near  the  temperature  of 
the  human  body.  This  is  not  necessarily  the  case  with  the 
non-pathogenic  species.  Ordinary  water  bacteria  thrive 
best  at  ordinary  temperatures. 

Sternberg's  method  for  determining  the  thermal  death-point 
of  a  species  of  bacteria  is  to  draw  a  portion  of  a  pure  culture  of 
.the  organism  to  be  tested  up  into  a  capillary  tube  which  has  a 
small  bulb  on  one  end,  and  after  sealing  the  end  of  the  capil- 
lary tube  in  the  flame  the  tubes  are  placed  upon  a  glass  plate 
in  a  water-bath,  whose  temperature  is  indicated  by  a  ther- 
mometer, while  a  uniform  temperature  is  secured  by  stirring. 


126  MANUAL    OF    BACTERIOLOGY. 

The  time  of  exposure  is,  as  a  rule,  ten  minutes.  The  tubes 
should  be  removed  quickly  to  cold  water.  Their  contents 
should  afterward  be  inoculated  into  bouillon  to  determine 
whether  or  not  the  organisms  have  been  killed.  In  the 
practical  use  of  heat  for  sterilization  or  disinfection,  the 
exact  thermal  death-point  is  greatly  exceeded.  The  time  of 
exposure  is  also  longer  than  is  absolutely  necessary  as  deter- 
mined by  the  results  of  the  experiments. 

It  is  hardly  safe  to  depend  upon  text-book  statements  in  re- 
gard to  the  thermal  death-point  of  bacteria  in  practical  disin- 
fection. 

Moisture  is  indispensable  to  the  growth  of  bacteria,  and  dry- 
ing causes  the  death  of  certain  kinds,  as,  for  instance,  the 
spirillum  of  cholera,  while  others  remain  alive,  but  do  not  grow. 

Heim*  found  that  the  resistance  of  organisms  to  drying  is 
very  much  greater  when  the  organism  in  question  is  contained 
in  the  pathological  material  from  animals  which  have  suc- 
cumbed to  the  disease,  on  the  one  hand,  than  when  it  is  derived 
from  cultures,  on  the  other.  The  pneumococcus  which  is  very 
sensitive  to  drying,  and  in  fact  is  difficult  to  keep  going  on 
culture  media,  remains  alive  for  16  months  and  preserves  its 
virulence  for  more  than  a  year  when  it  is  contained  in  blood 
from  an  animal  which  has  died  of  the  infection  dried  on  silk 
threads  and  kept  in  a  desiccator  containing  calcium  chloride. 
Similar  results  were  obtained  with  other  organisms. 

Food. — There  are  a  few  species  of  bacteria  that  contain 
chlorophyll,  but  it  is  wanting  in  most  forms.  On  account  of 
the  absence  of  chlorophyll,  bacteria  require,  as  part  of  their 
food,  organic  compounds,  such  as  sugar,  as  a  source  of  carbon. 
They  are  unable,  with  very  few  exceptions,  such  as  the  nitrify- 
ing bacteria,  to  derive  their  carbon  from  the  carbon  dioxide 
of  the  atmosphere,  or  from  inorganic  carbon  compounds.  Al- 
though some  species  are  able  to  obtain  nitrogen  from  inorganic 

*Zeitschr.  }.  Hygiene.     Bd.  L.,  No.  i,  April  4,  1905,  p.  123. 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.  127 

salts,  most  bacteria  flourish  best  if  organic  substances  contain- 
ing nitrogen,  like  peptone  and  albumen,  are  furnished  them  as 
part  of  their  food.  The  complicated,  unstable,  organic  mole- 
cules with  high  potential  energy  are  converted  by  them  into 
simple  and  more  stable  compounds  like  carbon  dioxide,  am- 
monia and  water,  with  the  liberation  of  energy.  These  facts 
become  manifest  in  connection  with  their  important  work  in 
decomposition,  putrefaction  and  fermentation.  A  culture- 
medium  having  a  slightly  alkaline  or  neutral  reaction  is  favor- 
able to  most  bacteria. 

The  prolonged  artificial  cultivation  of  bacteria  may  or  may 
not  modify  their  properties.  The  pathogenic  bacteria  are 
likely  to  undergo  considerable  modification  both  in  the  quality 
and  luxuriance  of  their  growth  and  the  intensity  of  their  path- 
ogenic characters. 

The  growth  of  bacteria  may  eventually  be  hindered  by  the 
accumulation  of  the  products  of  their  own  metabolism.  Many 
bacteria  refuse  to  grow  on. culture-media  at  all;  at  least  the 
suitable  artificial  medium  has  n6t  yet  been  found  for  them. 
Some  species  are  extremely  fastidious,  and  can  only  be  propa- 
gated on  particular  nutrient  substances,  others  again  will  grow 
in  distilled  water.*  But  bacteria  show  great  adaptability,  and, 
once  they  have  been  made  to  start,  they  can  be  further  culti- 
vated with  less  and  less  difficulty,  as  a  rule. 

Relation  to  Oxygen. — Oxygen  is  indispensable  to  the 
growth  of  bacteria.  Some  of  them,  the  aerobes,  require 
oxygen  in  the  free  form.  Others,  the  anaerobes  require  it  in 
some  form  of  compound,  and  are  unable  to  live  in  an  atmos- 
phere of  free  oxygen.  Others  still  are  able  to  flourish  either  in 
the  presence  or  absence  of  free  oxygen-facultative  aerobes  or 
anaerobes.  The  first-named  varieties  are  sometimes  called 
strict,  or  obligate  aerobes  or  anaerobes. 

*Bolton.     Zcitschrift  fur  Hygiene.     Ed.  I.,  No.  i,  1886. 


CHAPTER  II. 
PRODUCTS  OF  THE  GROWTH  OF  BACTERIA. 

THE  splitting  up  of  animal  and  vegetable  matter  by  the  bac- 
teria results  in  the  formation  of  various  products,  which  may 
possess  certain  characteristics.  Thus  some  of  the  products 
of  bacterial  growth  are  phosphorescent,  some  are  marked  by 
more  or  less  vivid  color,  others  again  by  poisonous  properties. 

Phosphorescence. — Bacteria  whose  cultures  exhibit  phos- 
phorescence have  been  found  in  the  ocean  and  in  fish. 

Chromogenic  Bacteria. — Many  bacterial  growths  display 
brilliant  coloring.  The  different  species  of  sarcinae  are  re- 
markable for  forming  highly-colored  growths;  some  of  them 
are  rose-red,  some  orange-yellow,  some  lemon-yellow,  and  so  on. 
The  bacillus  prodigiosus  presents  a  brilliant  red  growth  whose 
rapid  development  is  said  to  have  formed  the  basis  for  the  so- 
called  "Miracle  of  the  Bleeding  Host"  (see  page  5).  The 
bacillus  pyocyaneus  in  culture  gives  a  brilliant  green  fluores- 
cence and  is  responsible  for  the  color  of  blue  or  green  pus. 
Bacilli  which  exhibit  a  green  fluorescence  in  cultures  are  com- 
mon in  water.  In  cultures  on  potato  or  agar  the  colors  of 
the  chromogenic  forms  are  usually  well  shown.  The  pig- 
ment formed  by  the  chromogenic  bacteria  is  not  produced  in 
the  cells  themselves.  These  are  colorless.  The  color  is  due 
to  substances  excreted  by  the  cells  or  formed  from  material  in 
the  culture-media. 

Ferments  or  Enzymes.* — Many  bacteria  form  ferments 
which  have  the  power  of  dissolving  proteid  substances  in  a 
manner  similar  to  trypsin.  The  liquefaction  of  gelatin  is  a 

*Consult  Buxton.     Mycotic  Anzymes.     American  Medicine.     July  25,  1903. 

128 


PRODUCTS    OF    THE    GROWTH    OF    BACTERIA.  129 

I  familiar  example  of  this  process.     The  property  of  liquefying 

?  gelatin,  or  the  failure  to  do  so,  is  used  in  classifying  bacteria 
and  in  determining  the  nature  of  unknown  species. 

Some  bacteria,  as  the  bacillus  coli  communis,  form  ferments 
which  act  like  rennet  in  coagulating  milk.     Other  bacteria  are 

i  capable  of  forming  sugar  from  starch.     Others  have  the  power 

|  of  changing  cane  -sugar  into  glucose. 

Bacteria  which  are  able  to  decompose  cellulose  are  found  in 

j  the  stomachs  of  ruminant  animals.  Although  it  is  doubtful 
whether  the  products  of  cellulose  decomposition  have  any 
nutritive  value,  the  process  is  probably  useful  in  effecting  a  sub- 
division of  the  coarse  food,  consisting  of  grass,  hay  and  the  like. 
Some  bacteria  have  the  power  of  decomposing  neutral  fats 
into  fatty  acids  and  glycerin,  after  the  manner  of  the  fat-split- 
ting ferment  of  the  pancreatic  juice. 

The  end-products  which  result  from  the  growth  of  bacteria 
upon  albuminous  nutrient  media  are  very  numerous.  They 
are  complicated  and  not  well  understood.  Among  these  end- 
products  may  be  mentioned  peptone,  indol,  skatol,  phenol, 
leucin  and  tyrosin.  Nearly  related  are  the  toxins  (see  Chapter 
VI.),  which  play  an  important -part  in  the  production  of  dis- 
ease by  pathogenic  bacteria.  In  the  decomposition  of  urine 
by  bacteria  the  urea  is  converted  into  ammonium  carbonate. 
The  formation  of  indol  in  cultures  is  an  important  peculiarity 
of  certain  bacteria.  The  manner  of  making  the  test  for  indol 
is  somewhat  differently  described  by  different  authors,  but  the 
Committee  on  Standard  Methods  of  Water  Analysis,  American 
Public  Health  Association,*  recommend  the  following: 

The  organism  to  be  tested  must  be  made  in  broth  from 
which  all  traces  of  muscle  sugar  have  been  removed  or  in 
peptone  broth.  The  cultures  must  be  incubated  at  37°  C.  for 
four  days.  In  applying  the  test  two  drops  of  concentrated 

^Journal  0}  Injections  Diseases.  Supplement  No.  i,  May,  1905,  p.  115. 
See  also  in  this  connection  Tobey.  Journ.  Med.  Research.  XV.,  1906,  p.  301. 

9 


130  MANUAL    OF    BACTERIOLOGY. 

sulphuric  acid  and  one  c.c.  of  a  o.oi  per  cent,  solution  of 
sodium  nitrite  are  added  to  the  culture  after  it  has  been  allowed 
to  grow  for  this  length  of  time,  and  after  this  addition  the 
culture  is  observed  at  the  end  of  one-half  hour.  The  ap- 
pearance of  a  pink  color  indicates  the  presence  of  indol. 

Another  method,  and  quite  a  delicate  one,  after  adding  the 
sodium  nitrite,  is  to  run  in  a  layer  of  sulphuric  acid  beneath  the 
culture  so  as  to  form  a  layer  of  acid  below  and  culture  above. 
The  presence  of  indol  is  indicated  by  a  pink  ring  at  the  point 
of  juncture  of  the  acid  and  the  culture.  If  the  reaction  is 
obtained  with  sulphuric  acid  alone  without  the  addition  of  the 
nitrite,  it  indicates  that  both  nitrites  and  indol  are  present  in 
the  culture.  Blank  tests  must  be  made  with  the  same  culture- 
medium  which  is  employed  for  the  cultures,  since,  as  was 
shown  by  Wherry,*  nitrates  and  probably  also  nitrites  may 
gain  entrance  to  artificial  culture,  media  from  various  sources, 
such  as  the  water,  peptone  and  filter-paper  used  in  preparing 
the  media.  Wherry  also  showed  that  a  sufficient  quantity  of  i 
nitrites  may  be  absorbed  from  the  air  of  the  laboratory  in  a 
few  days  to  yield  a  distinct  indol  reaction. 

Rivasf  found  that  a  much  more  delicate  test  than  the  above 
consists  in  the  use  of  i  c.c.  of  a  10  per  cent,  solution  of 
sodium  hydroxide  in  place  of  the  sodium  nitrite,  and  the  use 
of  i  c.c:  of  50  per  cent,  sulphuric  acid.  These  are  mixed 
with  the  culture  to  be  tested.  This  test  gives  a  bright  purple 
or  pinkish  coloration  with  outlines  of  B.  coli,  but  not  with  its 
congeners. 

Check  blank  tests  are  especially  demanded  where  the  culture- 
medium  has  been  prepared  by  growing  Bacillus  coli  in  the 
meat  infusion  to  free  it  from  sugar,  since  Bacillus  coli  itself 
forms  indol  in  appreciable  amounts. 

The  Committee  on  Methods  of  Identification  of  Bacterial; 

*Wherry.     Journal  of  Infectious  Diseases.     Vol.  2,  1905,  pp.  436-44^. 
•\Journ~ Infec.  Dis.     Vol.  IV.,  No.  4,  Nov.  15,  1907.     pp.  642-646.' 

, 


PRODUCTS    OF    THE    GROWTH    OF    BACTERIA.  131 

Species,  Society  of  American  Bacteriologists,  recommends  that 
ammonia  and  indol  tests  be  made  on  cultures  at  the  end  of  the 
tenth  day,  nitrite  tests  at  end  of  the  fifth  day  (see  Appendix  I., 
Note  5). 

The  reaction  may  be  hastened  by  warming  slightly.  The 
value  of  this  reaction  will  be  understood  when,  to  give  one 
illustration,  it  is  remembered  that  the  bacillus  coli  communis 
usually,  if  not  always,  produces  indol  and  the  bacillus  of 
typhoid  fever  usually  does  not,  except  in  cultures  kept  for 
some  time — ten  days  or  more.  Morris*  found  that  in  cultures 
of  B.  typhosus,  cultivated  at  37  °  C.  for  this  length  of  time, 
indol  was  always  present,  but  not  in  cultures  of  this  organism 
which  were  one  or  two  days  old.  The  recommendation 
of  the  Society  of  American  Bacteriologists  that  the  indol  test 
should  be  made  in  cultures  ten  days  old  would  seem  not  to 
apply  for  differentiation  of  B.  typhosus.  The  reaction  depends 
upon  the  liberation  of  nitrous  acid  which,  with  indol,  forms 
a  red  color. 

The  change  of  organic  substances  into  more  stable  ones  does 
not  cease  with  the  compounds  mentioned  above.  Certain 
bacteria  of  the  soil,  which  will  be  discussed  further  on,  are  able 
to  complete  the  conversion  of  ammonia  into  nitrous  acid,  lead- 
ing to  the  formation  of  nitrites;  and  others  still  that  of  nitrites 
into  nitric  acid,  which  at  once  forms  nitrates. 

Formation  of  Acids. — In  the  course  of  their  growth  many 
bacteria  produce  acids,  especially  from  substances  containing 
sugar.  The  power  of  developing  lactic  acid  is  possessed  by  a 
large  number  of  species.  Acetic  acid  is  another  common  by- 
product. Besides  these,  butyric  acid,  formic  acid,  propionic 
acid  and  many  more  are  formed  by  different  bacteria. 

Development  of  Gas. — The  evolution  of  gas  from  bacterial 
growths  is  of  frequent  occurrence.  Many  bacteria  have  the 

*Cited  from  Giinther.  Einfiihrung  in  das  Studium  der  Bakteriologie.  6te 
Auf.,  1906,  p.  526. 


132 


MANUAL    OF    BACTERIOLOGY. 


property  of  splitting  up  organic  compounds  with  the  formation 
of  various  gaseous  products.  Perkins*  has  found  that  the 
power  of  thus  breaking  up  sugars  with  the  formation  of  gas- 
eous products  may  be  lost  in  whole  or  in  part  by  modification 
of  environment.  In  some  cases  the  power  would  seern  to  be 
permanently  lost,  in  others  it  may  be  recovered  again.  This 
observation  makes  it  necessary  to  interpret  with  great  caution 
the  results  of  fermentation  tests  as  a  specific  means  of  differen- 
tiation between  organisms  -  which 
are  otherwise  alike.  It  is  important 
to  bear  this  fact  in  mind  particularly 
in  the  diagnosis  of  the  members  of 
the  colon  group.  Carbon  dioxide, 
hydrogen  sulphide  and  nitrogen  are 
among  the  better  known  gases  that 
may  be  formed.  The  odors  that 
arise  from  cultures  and  that  are  so 
characteristic  of  putrefactive  pro- 
cesses depend  upon  the  develop- 
ment of  gases,  or  a  mixture  of 
gases,  of  considerable  complexity. 
The  bacillus  aerogenes  capsulatus 
leads  sometimes  to  the  formation 
of  gas  in  the  organs  of  the  human 
cadaver  within  a  short  time  after 
death.  Theobald  Smith  intro- 
duced a  valuable  means  of  differentiating  species  of  bacteria 
based  upon  their  power  of  forming  gas  in  media  containing 
different  sugars,  or  in  their  inability  to  do  so.  Bouillon  con- 
taining i  per  cent,  of  dextrose  (or  lactose,  etc.)  is  the  culture- 
medium  advised.  The  test  is  best  conducted  in  a  U-shaped 
tube,  closed  at  one  end,  and  at  the  other  end  provided  with  a 
bulb  (Fig.  48).  The  tube  is  stoppered  with  cotton,  sterilized 

*Perkins.     Journ.  Infect.  Dis.     Vol.  4    No.  i,  pp.  51-65. 


FIG.  48. — Fermentation-tube. 


PRODUCTS    OF    THE    GROWTH    OF   BACTERIA.  133 

by  dry  heat,  afterward  filled  with  the  bouillon,  and  sterilized 
by  steam  in  the  usual  manner.  After  the  last  sterilization  it 
;  should  be  tilted  until  the  closed  end  is  completely  filled  with  the 
medium.  After  it  has  been  inoculated  with  the  species  under 
consideration,  any  development  of  gas  will  be  indicated  by  the 
collection  of  the  gas  at  the  closed  end.  The  amount  of  gas 
formed  may  be  estimated  and  its  quality  tested.  To  accomp- 
lish the  latter  fill  the  bulb  with  2  per  cent,  solution  of  sodium 
hydroxide,  close  the  outlet,  and  tilt  the  tube  to  allow  the 
mixture  to  come  in  contact  with  the  gas.  After  shaking,  this 
causes  the  absorption  of  the  carbon  dioxide  and  diminution  in 
the  quantity  of  gas.  The  portions  which  remain  may  be  mixed 
with  air  and  ignited,  when  the  presence  of  hydrogen  and  some 
of  its  compounds  will  be  indicated  by  an  explosion.  (See  The 
Detection  of  Bacillus  coli  communis  in  Water,  Part  IV.) 

The  development  of  gas  may  readily  be  tested  by  inoculating 
the  bacteria  by  a  deep  puncture  into  agar  containing  i  per  cent, 
of  dextrose  or  other  sugars.  The  development  of  gas  causes 
bubbles  to  form  in  the  agar,  often  to  the  extent  of  splitting  it, 
and- sometimes  forcing  out  the  cotton  plug  (see  Fig.  73). 

The  activities  of  bacteria  which  have  just  been  enumerated 
are  fundamental  to  the  phenomena  which  go  by  the  names  of 
fermentation  and  putrefaction.  These  words  have  been  defined 
differently  at  different  times  and  by  different  writers,  but  in 
general  both  are  used  as  names  for  the  breaking  up  of  complex 
organic  compounds  by  micro-organisms  with  the  formation  of 
simpler  compounds.  Fermentation  refers  especially  to  the 
formation  of  useful  products  like  alcohol.  The  term  putre- 
faction is  employed  chiefly  for  the  breaking  up  of  nitrogenous 
compounds  with  the  development  of  foul-smelling  gases.  The 
term  fermentation  is  also  applied  to  the  decomposition  of  com- 
plex substances  through  the  influence  of  unorganized  ferments 
or  enzymes.  The  work  of  bacteria  in  decomposition  is  indis- 
pensable to  the  existence  of  the  organic  world  as  'we  find  it. 


134  MANUAL    OF    BACTERIOLOGY. 

Green  plants  convert  the  stable  compounds  of  nitrogen,  the 
carbon  dioxide  of  the  atmosphere  and  water  into  the  complej 
and  unstable  albumens  and  carbohydrates  which  serve  as 
food  for  animals.  Animals,  on  the  other  hand,  convert  these 
unstable  and  complex  compounds  back  into  simpler  forms, 
The  work  of  changing  them  back  into  the  simple  and  stable 
condition,  in  which  they  serve  as  the  food  for  plants,  is  per- 
formed by  animal  life  in  part  only,  and  its  completion  is  left  tc 
the  activities  of  bacteria.  It  is  the  work  of  bacteria  in  this 
direction  which  we  call  decomposition.  Without  that  work  the 
existence  of  life  upon  the  earth,  as  we  understand  it,  would  soon 
come  to  an  end,  and  the  dead  and  undecomposed  bodies  oi 
living  things  and  their  products  of  all  kinds  would  lie  about 
unchanged,  as  they  had  fallen. 

Bacterium  termo  is  the  name  formerly  given  to  a  supposed 
species  of  bacteria  which  was  credited  with  being  the  producei 
of  putrefaction.  The  individuals  were  represented  as  bein£ 
short  rods,  mostly  growing  in  pairs,  and  actively  motile.  The 
term  has  been  abandoned  since  it  appears  to  have  included  i 
number  of  different  species. 


CHAPTER  III. 

THE   BACTERIA  OF  SOIL,  AIR,  WATER,  AND  OF 
MILK  AND  OTHER  FOODS. 

The  Bacteria  of  the  Soil. — Bacteria  are  present  in  the  soil 
in  enormous  numbers— 100,000  or  more  in  i  c.c.  of  virgin  soil, 
according  to  Fliigge.  The  depths  to  which  they  penetrate 
depend  upon  the  character  of  the  soil  and  the  character  of  the 
life  upon  it,  and  whether  or  not  it  has  been  artificially  dis- 
turbed, as  by  cultivation.  In  general,  at  a  depth  of  1.25  meters 
(about  four  feet)  the  number  becomes  very  small,  and  at 
a  depth  of  a  few  decimeters  more  the  soil  may  be  found  en- 
tirely sterile.* 

The  bacilli  of  tetanus  and  malignant  edema,  and  bacillus 
aerogenes  capsulatus  are  present  in  the  soil  of  many  localities. 
According  to  Woodhead,  certain  savage  tribes  of  Africa  and 
the  East  Indies  use  as  an  arrow-poison  soil  that  is  capable  of 
producing  tetanus.  The  bacillus  of  anthrax  may  be  found  in 
soil  which  has  been  infected  with  this  organism. 

Most  of  the  bacteria  of  the  soil  are  harmless  and  some  of 
them  are  useful  saprophytes.!  To  the  latter  class  belong  the 
nitrifying  bacteria  described  by  Winogradsky  and  by  Jordan 
and  Richards  and  those  organisms  occurring  in  soil  which 
have  the  power  of  converting  ammonia  into  nitrous  acid  which 
forms  nitrites,  and  others  which  complete  the  change  of  nitrites 
into  nitrates.  Both  kinds  are  widely  distributed.  These 
organisms  will  not  grow  on  ordinary  culture-media,  and  their 
cultivation  presents  great  difficulties.  Probably  a  good  many 

*  Giither,  Soc.  cit.  p.  291.     See  also  Voorhees  and  Lipman.     W.  S.  Dept.  of 
Agr.Exp.  Stat.  Bulletin  194.     Oct.  26,  1907. 
t  See  Conn.  Agricultural  Bacteriology. 

135 


136  MANUAL   OF   BACTERIOLOGY. 

bacteria  have  similar  properties  to  some  extent.  The  work 
done  by  nitrifying  bacteria  in  the  formation  of  nitrates  from 
sewage,  manure  and  the  like  is  indispensable  to  most  plant 
life.  Certain  bacteria  found  in  the  soil  are  also  concerned  in 
the  assimilation  of  free,  atmosphere  nitrogen,  resulting  in  the 
addition  of  a  valuable  proportion  of  nitrogen  compounds  to  the 
soil.  This  is  spoken  of  as  nitrogen  fixation.  Inasmuch  as  a 
large  part  of  the  excrementitious  products  of  animals  contain- 
ing nitrogen  are  not  retained  in  the  soil,  where  they  may  be 
employed  as  food  by  plants,  but  are  washed  directly  or  in- 
directly into  the  sea  by  means  of  sewage  and  the  rivers,  it  will 
be  seen  that  the  supply  of  nitrogen  compounds  might  suffer 
gradual  exhaustion.  Furthermore,  it  has  already  been  noticed 
(page  131)  that  one  of  the  products  of  decomposition  by  bac- 
teria is  nitrogen,  which  is  not  available  as  food  for  animals  or 
for  most  plants.  These  facts  have  met  with  practical  recogni- 
tion by  agriculturists  in  the  adoption  of  various  methods  of 
fertilizing  the  soil.  It  appears  that  the  roots  of  peas,  beans, 
clover,  alfalfa  and  some  other  plants  frequently  present  minute 
tubercles  which  are  caused  by  the  development  of  microor- 
ganisms related  to  the  bacteria.  These  organisms  appear  to 
have  the  power  of  assimilating  atmospheric  nitrogen  and  of 
converting  it  into  nitrogen  compounds.  The  same  property 
probably  belongs  to  some  other  bacteria  of  the  soil.  Experi- 
ments show  that  these  observations  may  be  destined  to  be  of 
great  value  to  the  farmer.* 

The  bacteria  of  the  soil  may  easily  be  studied  in  plate- 
cultures  made  from  small  portions  of  soil  collected  with  the 
necessary  precautions  to  avoid  contamination,  or  plate-cultures 
may  be  made  from  sterilized  water  with  which  a  portion  of  the 
soil  has  been  properly  mixed.  Anaerobic  bacteria  must  be 
cultivated  by  the  special  methods  adapted  to  them. 

*For  practical  application  to  agriculture  consult  G.  Moore.  U.  S.  Dept. 
Agriculture.  Bureau  of  Plant  Industry,  Bulletin  No.  71,  Jan.  23,  1905.  See 
also  various  Bulletins  of  the  Department  of  Agriculture  on  this  subject. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  137 

Bacteria  of  the  Air. — The  bacteria  of  the  air  will  be  found 
for  the  most  part  clinging  to  solid  particles  in  suspension  in  the 
shape  of  dust  also,  as  shown  by  Fliigge,  to  particles  of  sputum 
thrown  out  by  efforts  in  coughing.  The  minute  air  bubbles 
thrown  off  in  this  way  remain  suspended  for  some  time.  Still, 
as  has  already  been  stated,  bacteria  cannot  be  blown  from 
moist  surfaces,  they  are  not  removed  by  currents  of  air.  Con- 
ditions of  dryness  and  wind  tend  to  increase  the  number  of 
microorganisms  in  the  air.  They  are  fewer  after  a  fall  of  rain 
or  snow,  and  the  number  is  smaller  in  winter  than  in  summer. 
The  air  of  cities  contains  more  bacteria  than  that  of  the 
country.  The  atmosphere  over  the  sea  and  at  the  tops  of  high 
mountains  is  nearly  or  wholly  free  from  bacteria.  The  bac- 
teria which  do  occur  in  the  air  are  seldom  pathogenic.  Their 
character  depends  upon  the  character  of  the  dust.  It  is  ob- 
vious that  dust  which  consists  in  part  of  the  dried,  pulverized 
expectoration  of  cases  of  pulmonary  tuberculosis  may  contain 
tubercle  bacilli.  Anthrax  of  the  lungs  sometimes  arise  in  men 
who  handle  the  wool  of  sheep  that  were  infected  with  anthrax 
(wool-sorter's  disease),  and  is  due  to  the  inhalation  of  anthrax 
spores  attached  to  the  wool.  The  atmosphere  in  the  im- 
mediate vicinity  of  cases  of  the  exanthematous  fevers  is  liable 
to  contain  the  organisms,  whatever  they  may  be,  that  cause 
these  diseases.  • 

In  a  rough  way,  one  may  obtain  some  knowledge  of  the 
character  of  the  oranisms  in  the  air  of  a  given  locality  by  re- 
moving the  cover  of  a  Petri  dish  containing  sterilized  gelatin 
or  agar  or  thin  slices  of  boiled  potato  for  a  few  minutes,  re- 
placing it,  and  allowing  the  organisms  to  develop.  In  most 
cases  a  large  proportion  of  the  growth  that  appears  will  be 
moulds.  Yeasts  are  also  common,  and  among  the  bacteria 
the  micrococci  are  abundant.  Chromogenic  varieties  are 
likely  to  be  present. 

A  few  studies  of  this  character  will  show  that  the  number  of 


138  MANUAL    OF    BACTERIOLOGY. 

organisms  that  are  present  depends  chiefly  upon  whether  the 
air  is  quiet  or  has  recently  been  disturbed  by 'draughts,  gusts 
of  wind  or  sweeping.  These  facts  are  of  fundamental  im- 
portance in  laboratory  work,  where  plate-cultures  are  being 
studied,  if  we  wish  to  avoid  contamination  of  the  plates. 
Among  various  devices  that  have  been  proposed  for  the  ac- 
curate study  of  the  organisms  of  the  air,  the  Sedgwick-Tucker 
aerobioscope  is  the  simplest  and  most  accurate.  It  consists 
of  a  glass -cylinder  (Fig.  49)  a  few  inches  long  and  an  inch  or  two 
in  caliber  with  a  narrow  neck  at  one  end  and  a  narrow  tube 
annealed  to  the  other.  A  layer  of  granulated  sugar  of  an 
inch  or  more  is  packed  loosely  in  the  narrow  tube,  and  the  neck 
and  the  end  of  the  narrow  tube  are  plugged  with  cotton.  The 


FIG.  49. — Sedgwick-Tucker  aerobioscope. 

instrument  is  sterilized  in  the  hot-air  sterilizer.  After  removing 
the  cotton  a  definite  quantity  of  air  is  to  be  aspirated  through 
the  large  end,  which  may  be  done  by  means  of  a  suction-pump 
applied  to  the  other  end,  or  by  siphoning  water  out  of  a  bottle 
the  upper  part  of  which  is  connected  with  the  end  of  the 
aerobioscope  by  means  of  a  rubber  tube.  The  sugar  acts  as  a 
filter  and  sifts  out  of  the  air  the  microorganisms  which  are  con- 
tained in  it.  Liquefied  gelatin  or  agar  in  sufficient  quantity 
is  introduced  into  the  large  end  of  the  instrument  by  means  of 
a  bent  funnel;  and,  after  replacing  the  cotton,  it  is  mixed  with 
the  sugar  which  dissolves.  The  culture-medium  is  spread 
around  the  inside  of  the  larger  portion  of  the  tube  after  the 
manner  of  an  Esmarch  roll-tube.  The  bacteria  which  are 
filtered  out  by  the  sugar  develop  as  so  many  colonies  upon  the 
solidified  medium. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  139 

Bacteria  of  Water  and  of  Ice. — The  water  of  rivers,  lakes 
and  the  ocean  always  contains  bacteria.  The  number  of 
organisms  varies  greatly  in  different  places  and  under  dif- 
ferent conditions.  The  number  of  different  species  found  in 
water  is  also  very  large.  Ground-water*  contains  few  or  no 
bacteria  under  normal  conditions,  and  is  therefore  suitable  for 
a  source  of  water-supply,  when  a  sufficient  amount  is  available. 
The  possibility  of  contamination  of  the  ground-water  from 
unusual  or  abnormal  conditions  should  always  be  eliminated 
before  it  is  taken  for  drinking-water.  Numerous  epidemics  of 
typhoid  fever  have  been  traced  to  contamination  of  wells. 
The  location  of  wells  with  reference  to  privy-vaults,  and  other 
possible  sources  of  contamination  should  be  chosen  with  the 
greatest  care. 

The  ordinary  bacteria  of  waterf  are  harmless, -as  far  as  is 
known.  Bad  ordors  and  tastes  in  drinking  water  that  is  not 
polluted  with  putrid  material  are  usually  due  to  minute  green 
plants  (algae).  J  The  diseases  most  commonly  disseminated 
by  water  are  typhoid  fever  and  Asiatic  cholera,  and  probably 
also  dysentery.  The  results  of  experiments  testing  the 
length  of  time  which  the  cholera  spirillum  and  the  typhoid 
bacillus  may  persist  in  water  are  conflicting.  Many  epidemics 
of  cholera  and  typhoid  have  been  traced  to  water  polluted  with 
the  discharges  from  cases  of  these  diseases. 

By  self -purification  of  water  is  meant  the  removal  through 
natural  processes  of  contaminating  organisms  such  as  might 
occur  from  the  discharge  of  sewage  into  it.  It  depends  upon 
the  sedimentation  of  the  contaminating  material,  in  the  form 

*Ground-water  is  the  water  which — originally  derived  from  rain  or  snow — 
sinks  through  superficial  porous  strata,  like  gravel,  and  collects  on  some  under- 
lying, impervious  bed  of  clay  or  rock. 

fSee  Fuller  and  Johnson  The  Classification  of  Water  Bacteria.  Journal 
o}  Experimental  Medicine.  Vol.  IV.,  p.  609.  Jordan.  Journal  of  Hygiene. 
Vol.  III.,  Jan.,  1903. 

|G.  T.  Moore.  Contamination  of  Water  Supplies  by  Alga?.  Yearbook 
U.  S.  Department  of  Agriculture.  1902. 


140  MANUAL   OF    BACTERIOLOGY. 

of  mud,  upon  the  growth  of  the  ordinary  water-plants  and  pro- 
tozoa, upon  the  exhaustion  of  the  food-supply  by  the  growth  of 
bacteria  themselves,  upon  the  destructive  influence  of  direct 
sunlight,  and  the  dilution  of  the  matter  added  with  a  large 
volume  of  water.*  It  is  not  usually  to  be  relied  upon  as  a 
means  of  freeing  the  water-supply  from  pathogenic  bacteria. 

Storage  of  Water. — When  water  is  kept  in  large  reservoirs, 
the  solid  particles  in  it,  including  bacteria,  tend  to  fall  to  the 
bottom.  The  number  of  bacteria  in  a  water-supply  may  be 
considerably  reduced  in  this  way  by  allowing  sedimentation  to 
take  place  and  using  the  upper  portion  of  the  water. 

Filtration. — Filtration  on  a  large  scale  has  been  more  com- 
monly in  use  in  the  cities  of  Europe  than  elsewhere  until  lately. 
But  filtration-plants  now  exist  in  several  cities  of  the  United 
States.  By  this  method  98  per  cent,  to  99-  per  cent,  of  the 
bacteria  in  water  may  be  removed. 

Slow  Sand  Filtration.^ — The  filter  consists  of  successive  layers 
of  stones,  coarse  and  fine  gravel.  The  uppermost  layers  are 
of  fine  sand.  The  whole  filter  is  from  i  to  2  meters  thick. 
The  sand  should  be  60  cm.  to  1.2  meters  in  thickness.  The 
accumulated  deposit  from  the  water  and  a  little  of  the  fine  sand 
must  be  removed  from  time  to  time,  but  the  layer  of  fine  sand 
must  never  be  allowed  to  become  less  than  30  cm.  in  thickness. 
The  first  water  coming  from  the  filter  is  discarded.  The 
actual  filtration  is  done  largely  by  the  slimy  sediment  which 
collects  on  the  surface  of  the  layer  of  fine  sand.  The  filter- 
beds  may  be  several  acres  in  extent,  and  in  cold  climates 
should  be  protected  by  arches  of  brick  or  storie.  They  re- 
quire renewal  occasionally.  This  kind  of  filtration  has  come 
largely  into  use  since  the  cholera  epidemic  of  1892-93,  and 
it  appears  to  be  very  effective.  It  is  often  advisable  to  use 

*See  Jordan.     Journal  of  Experimental  Medicine.     Vol.  V.,  p.  271. 
fFor  a  full  discussion  see  Journal  American  Medical  Association.     Oct.  3,  to 
3i,  1903- 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  141 

storage  basins  in  connection  with  sand  nitration,  to  permit  of 
settling  of  part  of  the  solid  matter  before  nitration. 

The  results  obtained  by  nitration  depend  greatly  upon  the 
intelligence  displayed  in  operation. 

Mechanical  Filtration. — This  method  of  nitration  is  also 
called  the  American  system.  It  is  more  rapid  than  the  pre- 
ceding method  and  does  not  require  a  large  area  for  filter  beds. 
Although  sand  is  required  also,  nitration  is  accomplished  by 
a  jelly-like  layer  of  aluminum  hydroxide.  This  product  is 
formed  by  adding  to  the  water  a  small  quantity  of  aluminum 
sulphate  or  of  alum.  The  carbonates  in  the  water  decompose 
the  aluminum  salt  and  produce  aluminum  hydroxide.  It 
precipitates  as  a  white,  flocculent  deposit,  entangling  solid  par- 
ticles, including  bacteria,  as  coffee  is  cleared  with  white  of  egg. 
Only  a  trace  of  aluminum  should  appear  in  the  water.  This 
method  of  nitration  has  not  been  tested  so  extensively  as  slow 
sand  filtration,  but  seems  likely  to  prove  efficient.  With  water, 
poor  in  carbonates,  these  may  have  to  be  added.* 

Whipple  and  Longleyt  found  that  the  efficacy  of  mechani- 
cal filters  with  the  addition  of  alum  depends  somewhat  upon 
the  character  of  the  alum.  They  find  that  the  alum  shall  be 
shown  by  analysis  to  contain  17  per  cent,  of  alumina  (Ai2 
03)  soluble  in  water,  and  of  this-  amount  at  least  5  per  cent, 
shall  be  in  excess  of  the  amount  necessary  theoretically  to 
combine  with  the  sulphuric  acid  present.  It  shall  not  contain 
more  than  i  per  cent  of  insoluble  substances,  and  shall  be 
free  from  extraneous  debris  of  all  kinds.  It  must  not  contain 
more  than  0.5  per  cent,  of  iron  (Fe2O3)  and  the  iron  shall  be 
preferably  in  the  ferrous  state. 

Various  methods  for  the  purification  of  water  by  means  of 
chemicals  have  been  proposed.  The  use  of  copper  sulphate 
to  disinfect  drinking  water  was  recommended  by  Moore 

*See    Fuller.     Journal   American   Medical   Association.     Oct.    31,    1903 
\Jowrn,  Inject.  Diseases.     Supplement  No.  2,  Feb.,  1906,  pp.  166-171 


142  MANUAL    OF    BACTERIOLOGY. 

and  Kellerman,*  and  various  investigators  tested  the  value 
of  their  recommendation. 

Clark  and  Gagef  came  to  the  conclusion  from  their  investi- 
gation that  the  treatment  of  water  with  copper  sulphate  or  the 
storing  of  water  in  copper  vessels  has  little  practical  value. 
Others  also  have  come  to  practically  the  same  conclusion. 
While  the  addition  of  copper  sulphate  is  of  use  in  preventing 
the  growth  of  the  algae,  which  sometimes  grows  so  abundant, 
as  to  choke  up  water  pipes,  is  of  benefit  in  this  direction,  the 
weight  of  evidence  appears  to  be  against  its  efficacy  for  purifying 
water  for  drinking  purposes.  The  use  of  ozone  for  the  puri- 
fication of  water  has  met  with  considerable  favor.  J 

The  filtration  of  water  on  a  small  scale,  as  is  ordinarily  done 
for  domestic  purposes,  is  generally  entirely  useless.  The  so- 
called  Pasteur  filter  of  unglazed  porcelain  is  effective  if  it  is 
properly  constructed  and  if  the  filter-tubes  are  sterilized  by 
heat  every  few  days — conditions  which  are  seldom  complied 
with.  Distillation  of  water  and  boiling  are  the  most  practical 
methods  for  sterilizing  drinking-water. 

Collection  of  Samples. — For  bacteriological  examination 
samples  from  the  water-supply  of  a  city  may  be  drawn  from 
the  faucet,  but  the  water  should  first  be  allowed  to  run  for  half 
an  hour  or  longer.  From  other  sources  the  supply  should 
be  collected  in  sterilized  tubes  or  bottles,  taking  care  to  avoid 
contamination.  Sternberg  bulbs  (see  Fig.  38)  will  be  found 
useful  for  small  samples.  These  samples  should  be  examined 
as  quickly  as  possible,  for  the  water  bacteria  increase  rapidly 
in  number  after  the  samples  have  been  collected.  When  trans- 
portation to  some  distance  is  unavoidable  the  samples  should 
be  packed  in  ice,  but  even  this  precaution  does  not  preserve 
the  original  bacteriological  condition  of  the  water  at  the  time 

*U.  S  .Dep.  Agriculture.  Bu.  Plant  Ind.  Bulletin  64,  1904. 
~\Journ.  Inf.  Diseases.  Sup.  No.  2,  Feb.,  1906,  pp.  175-204. 
^Consult  Rosenau.  Disinfection  and  Disinfectants.  1902. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  143 

it  is  caught;  for  more  or  less  change  probably  takes  place  at 
all  temperatures.  If  the  temperature  is  too  high,  and  the 
water  freezes,  more  or  less  of  the  bacteria  may  be  killed;  if,  on 
the  contrary,  the  temperature  is  not  low  enough  there  will  be 
a  multiplication  of  the  bacteria  in  transit.  The  value  of  exam- 
ination of  water  shipped  in  any  way  is  of,  at  least,  doubtful 
utility. 

The  number  of  bacteria  may  be  determined  by  making 
plates  of  a  definite  quantity  of  the  water  with  gelatin  or  agar.* 
The  amount  examined  ordinarily  is  i  c.c.  When  the  number 
of  bacteria  is  very  large,  a  smaller  quantity  must  be  taken,  and 
it  may  be  necessary  to  dilute  the  sample  ten  times  or  more  with 
sterilized  water.  The  amount  should  be  measured  with  a 
sterilized,  graduated  pipette.  The  water  is  mixed  with  melted 
gelatin  or  agar  in  a  tube  which  has  been  allowed  to  cool  after 
melting.  After  thorough  mixing,  remove  the  plug,  burn  the 
edge  of  the  tube  in  the  flame,  hold  in  a  nearly  horizontal  position 
until  cool  and  pour  into  a  sterilized  Petri  dish.  Or  better, 
measure  the  water  into  the  Petri  dish,  and  pour  the  melted 
medium  in,  and  mix.  The  number  of  colonies  may  be 
counted  on  the  third  or  fourth  day;  the  later  the  better, 
as  some  forms  develop  slowly  and  may  not  present  visible 
colonies  for  several  days;  but  the  plates  are  often  spoiled 
after  three  or  four  days  by  the  profuse  surface  growths  of  cer- 
tain forms  or  by  the  rapid  liquefaction  of  gelatin,  if  that  be 
used,  by  other  forms.  The  number  of  colonies  that  develop  is 
supposed  to  represent  the  number  of  individual  bacteria  con- 
tained in  the  quantity  measured.  That  will  probably  not  al- 
ways be  the  case,  however,  as  colonies  may  develop  from  a 
clump  of  bacteria  which  have  not  been  separated  from  one 
another  by  the  mixing  process.  Abbott  has  shown  that  the 
number  of  colonies  is  usually  larger  on  gelatin  plates  than  upon 
agar  plates,  and  at  the  room  temperature  than  in  the  incubator. 

*For  preparation  of  culture  media  for  water  analysis,  see  p.  71. 


T44  MANUAL    OF    BACTERIOLOGY. 

This  observation  illustrates  the  fact  that  there  are  doubtless 
many  kinds  of  bacteria  that  do  not  find  favorable  conditions 
for  development  on  ordinary  culture-media.  The  reaction  of 
the  medium  has  an  important  influence  upon  the  develop- 
ment of  these  water  bacteria  in  plate  cultures. 

When  the  number  of  colonies  is  small,  there  is  no  difficulty 
in  counting  them  as  they  appear  in  the  ordinary  Petri  dish. 
When  the  number  is  large,  some  kind  of  mechanical  device 
may  be  used  to  assist  counting.  The  Wolffhiigel  plate  is  a 
large  square  of  glass  resting  in  a  wooden  frame  painted  black. 
The  glass  plate  is  ruled  in  squares.  It  was  designed  particularly 
with  reference  to  the  form  of  plate-cultures  first  made  by  Koch. 
The  Petri  dish,  however,  may  be  placed  upon  the  glass  plate 
and  the  cross  lines  be  used  to  assist  in  counting.  Lafar, 
Pakes  and  Jeffer  recommend  a  surface  painted  black,  ruled 
with  white  lines  which  represent  the  radii  of  a  circle,  which  may 
be  still  further  subdivided  by  other  lines.  Many  find  counting 
easier  when  a  black  surface  divided  into  squares  is  employed. 
An  ordinary  card  with  a  smooth  black  surface  divided  into 
squares  by  white  lines  may  be  placed  under  a  Petri  dish  and  will 
be  found  to  serve  very  well.*  For  the  mere  examination  of  the 
colonies  no  better  surface  can  be  devised  than  the  ferrotype 
plate  used  by  photographers.  The  examination  of  the  colonies 
will  be  easier  if  a  small  hand-lens  be  used.  Care  must  be 
taken  not  to  mistake  air-bubbles  or  particles  of  dirt  for  colonies 
of  bacteria. 

In  any  case,  if  possible,  all  the  colonies  in  the  plate  should  be  I 
counted.  But  if  this  is  not  possible,  the  number  contained  [ 
within  several  squares  may  be  counted  and  the  average  taken;  | 
knowing  the  size  of  the  squares  and  the  area  of  the  plate,  the  ; 
number  contained  in  the  whole  plate  may  be  calculated. 

The  plating  may  be  done  by  rolling  the  medium  after  the 

*Specially  ruled  cards  will  be  found  after  the  Appendix  II.,  at  the  back  of  the 
book. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  145 

manner  of  Esmarch.  When  the  number  of  colonies  is  not 
large  this  may  serve  very  well.  Counting  may  be  assisted 
by  drawing  lines  with  ink  on  the  outer  surface  of  the  test-tube. 
It  is  obvious  that  the  character  of  the  bacteria  is  of  prime 
importance;  that  pathogenic  organisms  may  occasionally 
be  present,  even  when  the  number  of  bacteria  to  the  cubic 
centimeter  is  small.  But  knowing  the  number  usually  found 
in  a  good  water-supply,  any  sudden  variation  above  that  num- 
ber is  to  be  looked  upon  with  suspicion  as  indicating  a  possible 
contamination. 

The  bacteriological  examination  should  always  be  accom- 
panied by  a  chemical  examination,  and  by  an  inspection  of 
the  surroundings.  A  large  number  is  to  be  expected  when 
the  water  has  been  subjected  to  unusual  agitation  from  winds 
or  currents  which  stir  up  the  bacteria  which  have  settled. 

The  detection  of  pathogenic  bacteria  in  water*  involves 
great  difficulties,  and  our  knowledge  in  this  direction  is  very 
meagre.  Koch  and  several  others  have  reported  finding  the 
spirillum  of  Asiatic  cholera  in  water.  The  examination  of 
water-supplies  for  this  organism  has  disclosed  the  fact  that 
bacteria  resembling  the  organism  of  cholera  in  many  respects 
are  not  uncommon  in  water.  This  adds  to  the  difficulty  of 
detecting  the  cholera  germ  in  water. 

The  bacillus  of  typhoid  fever  has  many  times  been  described 
as  occurring  in  water-supplies  suspected  of  being  contaminated 
with  the  excreta  of  cases  of  the  disease.  The  interpretation 
of  these  observations  is  at  present  doubtful. f  It  is  now  known 
that  several  forms  of  bacteria  exist  which  closely  resemble  the 
bacillus  of  typhoid  fever,  and  which  make  its  recognition  in  an 
unknown  specimen  very  difficult.  J 

It  will  at  once  be  appreciated  that  the  number  of  cholera  and 

*  See  also  articles  in  Part  IV.  on  the  bacillus  of  typhoid  fever,  bacillus  coli 
communis  and  spirillum  of  cholera. 

fConsult  editorial.     Journal  American  Medical  Association.     Dec.  5,  1903. 
£For  methods  of  detection  see  under  Typhoid  Bacillus,  p.  370. 


146  MANUAL    OF    BACTERIOLOGY. 

typhoid  organisms  necessary  to  contaminate  a  considerable 
body  of  water,  and  sufficient  to  cause  an  outbreak  of  the  dis- 
ease among  some  of  the  people  drinking  the  water,  may  still 
be  so  small  that  many  different  cubic  centimeters  of  the 
water  might  be  studied  before  a  single  one  of  the  specific  organ- 
isms would  be  encountered.  Anyone  who  has  examined  plates 
made  from  samples  of  water  will  recognize  the  difficulty  of 
detecting  one  or  a  few  colonies  of  the  bacteria  of  cholera  or 
typhoid  fever  among  a  hundred  or  more  colonies  of  ordinary 
water-bacteria.  The  existence  of  contamination  with  animal 
excreta  might,  however,  be  indicated  by  finding  the  bacillus 
coli  communis,  whose  detection  offers  a  greater  prospect  of 
success,  the  presence  of  small  numbers  of  the  colon  bacillus  in 
water  is  regarded  as  of  little  or  no  significance.*  Until  our 
knowledge  is  more  complete,  any  suspicious  water  should  be 
discarded. 

Formerly  investigators  seem  to  agree  that  if,  using  several  samples  of  a  water 
each  i  c.c.  in  volume,  colon  bacilli  are  found  in  a  majority  of  the  samples  the 
water  is  probably  polluted;  if  the  colon  bacillus  is  only  found  when  larger  volumes 
of  water  are  examined,  the  results  are  suspicious  though  less  significant.  Some 
investigators  hold  that  the  presence  of  streptococci  in  water  is  indicative  of  pollu- 
tion, f  At  present  there  seems  less  agreement  upon  these  points.  Johnson  $ 
found  that  the  colon  bacillus  is  ingested  by  fish  when  this  organism  is  present 
in  the  water  in  which  the  fish  are  kept,  and  that  the  bacillus  lives  and  multi- 
plies in  the  intestines  of  the  fish.  He  concludes  that  in  this  way  fish  may 
convey  the  colon  bacillus,  and  if  so  also  the  typhoid  bacillus,  from  a  contami- 
nated source  to  an  uncontaminated  stream. 

Certain  devices  have  been  adopted  to  hasten  the  develop- 
ment of  the  bacteria  indicative  of  pollution  and  to  retard  that 
of  the  ordinary  water-bacteria.  Among  these  may  be  men- 
tioned the  influence  of  the  heat  of  the  incubator,  which  will 
hasten  the  growth  of  organisms  derived  from  the  human  body, 
and  which  retards  the  growth  of  water-bacteria.  Another 

*Jordan.    Jaurn.  Am.  Med.  Assn.  V.  XLVIIL,  No.  22.  June  i,  1907,  p.  1861. 
fPrescott  and  Baker.     Journal  o}  Infectious  Diseases.  I.     193. 
%Journ.  Infect.  Diseases.     Vol.  I.,  pp.  348-354.' 


THE   BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  147 

is  the  addition  of  a  solution  of  peptone  to  a  large  quantity  of 
the  water  to  be  examined  with  a  view  to  assisting  the  develop- 
ment of  the  desired  bacteria  by  furnishing  them  suitable  food 
for  growth.  In  another  method  (Parietti's)  small  quantities  of 
carbolic  acid  are  added  to  bouillon  and  mixed  with  the  water, 
with  a  view  to  retarding  the  development  of  all  except  typhoid  * 
and  colon  bacilli;  Jacksonj  advocates  the  use  of  ox-bile  for 
the  same  purpose.  The  committee  on  Standard  Methods  of 
Water  Analysis,.  Am.  pub.  Health  Assn.,  J  suggest  the  follow- 
ing modifications  of  previous  recommendations  for  the  detec- 
tion of  B.  coli  in  water: 

Omit  determinations  of  motility:  omit  tests  for  coagulation 
of  milk  in  case  cultures  have  been  isolated  in  lactose  agar 
plates;  omit  determination  of  the  amount  of  gas  formed  and 
the  gas  ratio  in  both  presumptive  and  in  confirmatory  tests; 
allowing  the  use  of  bile-broth  instead  of  dextrose-broth. 
Suspected  bacteria  may  be  tested  by  inoculation  into  animals; 
the  possession  of  pathogenic  properties  is  thought  by  some  to 
create  a  probability  in  favor  of  their  having  come  from  some 
contamination  with  animal  excreta.  § 

If  it  is  not  already  apparent  from  what  has  been  said,  it  must 
be  here  emphasized  that  the  difficulty  of  detecting  the  presence 
of  pathogenic  bacteria  in  water  is  very  great,  and  the  length  of 
time  necessarily  consumed  in  making  the  tests  greatly  lessens 
the  value  of  the  results  when  obtained.  Added  to  this  is  the 
further  limitation  of  the  value  that  a  negative  result,  i.  e.,  where 
no  pathogenic  bacteria  found,  cannot  be  taken  as  proof  that 
the  water-supply  under  examination  may  not  be  contaminated 

*Prescott.  Report  of  American  Public  Health  Association.  Vol.  XXIX., 
356.  Clark  and  Gage.  Ibid.  386.  Bissell.  Ibid.  360. 

^Journ.  Infect.  Dis.,  Sup.  No.  3.     May,  1907.     p.  30-32. 

^American  Journ.  oj  Public  Hygiene.     Vol.  XVII.,  No.  4,  Nov.,  1907,  p.  367. 

§Consult  Vaughan.  Journal  American  Medical  Association.  April  9,  1904. 
For  special  methods  of  detecting  the  Bacillus  coli  communis  see  under  this 
bacillus,  page  310.  For  general  subject  of  Water  Supply  and  Public  Health 
see  Jordan.  Journ.  Am.  Med.  Assn.,  V.  XLVIIL,  May  i8-June  8,  1907. 


148  MANUAL   OF    BACTERIOLOGY. 

at  times.*  Fliiggef  has  shown  that  the  chemical  examination 
also  permits  of  no  conclusion  of  itself  as  to  the  potability  of  water 
It  would  seem  that  those  best  suited  by  training  and  experience 
and  who  are  capable  of  forming  disinterested  opinion  attach 
but  limited  importance  to  the  result  of  laboratory  examinations 
of  water  unaccompanied  by  a  sanitary  inspection.  Opinions 
based  upon  analyses  of  water  shipped  to  a  chemical  or  bacterio- 
logical laboratory  should  be  taken  with  reserve.  In  fact,  many 
of  those  who  have  made  disinterested  study  of  the  subject  are 
inclined  to  question  the  value  of  chemical  and  bacteriological 
water  analysis  in  toto,  and  in  view  of  the  arbitrary  and  mechani- 
cal manner  in  which  the  results  of  these  analyses  are  sometimes 
interpreted,  this  attitude  is  justified.  It  would  seem,  however, 
that  after  the  establishment  of  normal  standards  for  a  given 
locality,  such  analyses  are  useful  if  they  are  checked  by  intelli- 
gent consideration  of  all  the  conditions  entering  into  the  case, 
but  no  hard  and  fast  rules  can  be  applied.  { 

Ice. — The  bacteriological  examination  of  ice  differs  in  no 
respect  from  that  of  water.  Although  development  may  be 
arrested,  the  vitality  of  bacteria  is  not  necessarily  impaired  by 
freezing.  Prudden  found  the  bacillus  of  typhoid  fever  alive 
in  ice  after  more  than  one  hundred  days.  However,  Sedg- 
wick  and  Winslow  have  stated  that  when  typhoid  bacilli  are 
frozen  in  water  the  majority  of  them  are  destroyed.  §  Never- 
theless, it  is  as  necessary  to  have  the  source  from  which  ice  is 
taken  as  carefully  scrutinized  as  that  of  the  water-supply,  es- 
pecially in  view  of  the  universal  habit  of  cooling  water  in  the 
summer  time  by  adding  ice  directly  to  the  water.  It  is  better 


*Gunther  loc.  cit.  283. 

fFliigge.     Zeitschrijt  fur  Hygiene.     Bd.  22,  1896,  pp.  445  et  seq. 

|  Bolton.  Sanitary  Water  Supplies  for  Dairy  Farms.  Public  Health  and 
Marine  Hospital  Service.  Bulletin  41,  February,  1908,  p.  534. 

§Clark.  Bacterial  Purification  of  mater  by  Freezing.  Reports  American 
Public  Health  Association.  Vol.  XXVII.  See  also  Hutchings  and  Wheeler. 
American  Journal  Medical  Sciences.  Vol.  CXXVL,  p.  680 


THE   BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  149 

to  cool  water  and  articles  of  food  by  surrounding  the  vessels 
containing  them  with  ice. 

Bacteria  of  Milk  and  Other  Foods.*— Of  the  different 
food  substances,  milk  is  probably  the  most  important  from  a 
bacteriological  point  of  view.  In  the  first  place,  most  other 
focds  are  cooked  before  eating.  Furthermore,  cow's  milk 
constitutes  the  principal  fcod  of  young  infants,  who  are  highly 
susceptible  to  certain  bacteria  and  to  substances  in  the  milk 
itself,  after  it  has  undergone  certain  alterations  due  to  bacteria. 
The  milk  of  the  healthy  cow  as  it  is  secreted  in  the  mammary 
gland  is  sterile;  however,  after  milking  the  cow  a  little  milk 
generally  remains  in  the  milk-ducts  and  in  the  lower  part  of 
the  teat  in  which  numerous  bacteria  will  have  developed 
before  the  next  milking-time.*  The  first  milk  obtained  at  a 
milking  should  therefore  be  discarded,  as  it  may  contain  an 
excessive  number  of  bacteria. 

In  examining  milk  for  bacteria  the  number  may  be  estimated 
by  precisely  the  same  technic  as  is  used  for  the  estimation 
of  the  number  of  bacteria  in  water.  The  milk  requires  to  be 
diluted  usually  more  than  water  for  the  reason  that  there  are 
a  great  many  more  bacteria  ordinarily  present  in  milk  than  in 
water,  and  consequently  a  very  small  amount  of  milk  has  to 
be  used,  so  small  that  it  cannot  be  accurately  measured  except 
by  diluting.  If  much  milk  is  introduced  into  the  culture 
medium  it  makes  the  latter  opaque. 

A  committee  of  the  American  Public  Health  Association! 
recommend,  among  other  things,  the  following:  Milk  should 
be  plated  within  four  hours  after  it  is  collected,  and  in  the  mean- 
time should  be  kept  below  40°  C.,  but  not  frozen.  Dilutions 
recommended  are  i-io,  i-ioo,  i-iooo,  1-10,000,  1-100,000, 

*See  Conn.  Bacteria  in  Milk  and  its  Products.  1903.  Russell.  Dairy 
Bacteriology.  1906.  yth  Ed. 

fPreliminary  statement  by  the  committee  on  standard  methods  of  bacterial 
milk  analysis.  American  Journal  of  Public  Hygiene.  Vol.  XVII.,  No.  4, 
November,  1907. 


150  MANUAL    OF    BACTERIOLOGY. 

1-1,000,000.  The  i-io,  dilution  should  be  prepared  by 
shaking  the  sample  twenty-five  times  and  taking  i  c.c.  and 
putting  it  into  9  c.c.  of  sterile  water.  The  i-ioo,  dilution  is 
prepared  in  the  same  way,  adding  i  c.c.  of  the  milk  to  99  c.c. 
of  sterile  water.  The  other  dilutions  are  prepared  from  the 
i-iooo  dilution.  The  plate  giving  from  40  to  400  colonies 
should  be  selected  for  counting.  All  the  colonies  on  the  plate 
should  be  counted  in  preference  to  counting  any  part  of  a 
plate  and  calculating  the  total  number.  The  culture  medium 
should  be  agar,  containing  i  per  cent,  agar,  and  it  should 
be  made  from  the  watery  extract  of  beef.  The  reaction  should 
be  +  1.5,*  American  Board  of  Health  Scale.  The  majority  of 
those  consulted  by  the  committee  count  the  colonies  after 
twenty-four  hours,  incubation  at  37°  C.  in  a  moist  incubatcr. 

As  a  routine  procedure,  in  cold  weather,  entirely  satisfac- 
tory results  may  be  obtained  by  taking  i  c.c.  of  the  milk  to  be 
examined  after  it  is  thoroughly  mixed,  and  putting  it  into  9  c.c. 
of  sterile  water,  and  taking  i  c.c.  of  this  solution  in  9  c.c.  of 
sterile  water.  Plates  made  from  this  dilution  with  i-io  c.c.  and 
with  i  c.c.  respectively  have  been  found  to  give  closely  corre- 
sponding results,  and  unless  the  milk  is  badly  contaminated  it 
is  always  possible  to  count  the  colonies  readily.  In  warm 
weather  and  in  the  case  of  cream,  a  third  or  even  a  fourth  dilu- 
tion should  be  made.  Where  the  milk  or  cream  are  mixed 
with  the  medium  in  the  tube,  the  resulting  colonies  are  apt  to  be 
more  uniformly  distributed  on  the  plate  than  where  the  milk  is 
put  into  the  Petri  dish  and  the  culture  medium  poured  in  after- 
ward. The  number  of  bacteria  remaining  in  the  test-tube 
in  the  former  method  of  proceedure  must  be  very  few  where 
the  medium  is  properly  fluid  when  it  is  poured.  The  ob- 
jection to  this  method  would  appear  to  be  purely  theoretical, 
and  counterbalanced  by  its  -advantages. 

Contamination  of  the  milk  may  occur  from  the  outer  surface 

*See  page  67. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  151 

of  the  udder  of  the  cow,  from  the  hands  of  the  milker,  from 
dirty  pails,  through  agitation  of  the  air  of  the  stable,  and  in 
other  ways.  In  view  of  these  sources  of  contamination  it  is 
not  to  be  wondered  at  that  the  number  of  bacteria  found  ordi- 
narily in  milk  is  usually  very  large.  In  ordinary  milk  as  fur- 
nished by  milkmen  the  number  of  bacteria  to  the  cubic  centi- 
meter is  usually  many  thousands,  and  may  run  up  to  many 
millions.  Milk  as  obtained  from  the  grocers,  where  it  is  apt 
not  to  be  kept  properly  cooled,  frequently  contains  hundreds 
of  thousands  or  many  millions  of  bacteria  to  the  cubic  centi- 
meter. Many  or  most  of  these  bacteria  must  be  little  if  at 
all  detrimental  to  health,  otherwise  milk  would  be  entirely 
unsuitable  as  fcod  in  the  raw  state;  whereas  this  is  not  the  case. 
On  the  contrary,  there  are  some  bacteria  which  are  useful  in 
the  manufacture  of  dairy  products.  The  ripening  of  cream 
and  of  cheese  is  due  to  the  growth  of  bacteria  introduced  in 
cultures  or,  what  amounts  to  the  same  thing,  it  is  due  to  the 
growth  of  bacteria  intrcduced  in  some  previously  ripened 
cream  or  cheese.  This  imparts  the  agreeable  flavor.  Moulds 
are  also  used  to  give  the  peculiar  flavor  to  some  cheeses.* 

But  aside  from  these  more  or  less  innocuous  forms  and 
those  which  are  useful,  milk  may  at  times  contain  pathogenic 
bacteria. 

Tuberculosis  is  a  disease  to  which  cattle  are  exceedingly  prone. 
There  is  good  reason  to  believe  that  infants  acquire  tuberculo- 
sis through  taking  as  feed  the  milk  of  tuberculous  cows,  al- 
though the  danger  from  this  source  has  probably  been  over- 
estimated. The  milk  of  tuberculous  cows  may  contain  tubercle 
bacilli  when  there  is  no  tuberculous  disease  of  the  udder. t 
The  frequency  of  tuberculosis  among  milch  cows  sometimes 
becomes  as  high  as  25  per  cent.,  or  even  more.  Butter  derived 

*Conn.     Agricultural  Bacteriology. 

fMohler.  Infectiveness  of  milk  of  cows  which  have  reacted  to  the  tuberculin 
test.  U.  S.  Department  of  Agriculture,  Bureau  of  Animal  Industry.  Bulle- 
tin No.  44,  1903. 


152 


MANUAL   OF    BACTERIOLOGY. 


from  the  milk  of  such  cows  may  contain  tubercle  bacilli.  The 
proper  manner  for  the  States  to  deal  with  this  problem,  for  it 
is  one  that  doubtless  will  fall  to  the  individual  States,  has  not 
yet  been  determined.  The  cost  of  killing  such  a  large  number 
of  valua  ble  cows  would  be  very  great.  Furthermore,  it  is  by 
no  means  certain  that  this  procedure  would  eradicate  the  dis- 
ease. The  flesh  of  cattle  also  is  capable  of  transmitting 
tuberculosis,  but  is  a  less  serious  source  of  danger  when  beef 
is  thoroughly  cooked. 

Epidemics  of  typhoid  fever  and  cases  of  diphtheria  have 
been  traced  to  milk;  and  no  doubt  cholera  asiatica  is  conveyed 
in  this  way  in  times  of  epidemic. 

Human  milk  often  contains  streptococcus  epidermidis 
albus,  and  not  seldom  the  staphylococciis  pyogenes  aureus 
under  normal  conditions. 

Scarlet  fever  is  probably  conveyed  by  milk,  but  as  the  organ- 
ism causing  this  disease  is  not  yet  definitely  known,  it  has  not 
yet  been  detected  in  milk.  But  there  is  apparently  good  clini- 
cal evidence  of  this. 

Streptococci  have  been  found  quite  frequently  in  milk  sold 
in  the  market.*  Bacillus  coli  communis  is  very  often  present 
in  milk,  but  it  is  probably  without  significance  unless  it  is 
present  in  very  large  numbers  when  it  is  possible  that  it  causes 
injurious  fermentative  changes.  Fermentative  changes  are 
also  caused  by  the  presence  of  bacteria  which  are  not  in  them- 
selves the  cause  of  disease,  and  these  changes  may  render  the 
milk  unfit  for  consumption  or  even  poisonous.  These  altera- 
tions may  be  evident  to  the  senses,  as  the  ordinary  lactic  acid 
fermentation  (souring  of  milk),  or  they  may  not.  The  charac- 
ter of  the  alterations  doubtless  varies  much  with  the  tempera- 
ture and  with  the  character  of  the  contaminating  bacteria. 
Summer  temperatures  of  course  favor  decomposition  and  fer- 

*Reed  and  Ward.  The  Significance  of  the  Presence  of  Streptococci  in  Market 
Milk.  American  Medicine.  February  14,  1903. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  153 

mentation.  Specialists  in  children's  diseases  attribute  to  al- 
terations in  milk  with  the  formation  of  poisonous  substances 
a  preeminent  influence  in  the  production  of  the  intestinal  dis- 
orders of  infancy  so  common  in  the  summer. 

Poisoning  with  milk,  ice-cream  or  cheese  is  not  rare,  as  is 
well  known.  There  are  many  records  of  whole  companies 
of  individuals  having  been  taken  violently  ill  after  having  eaten 
one  of  these  feeds  from  the  same  source  of  supply.  The  symp- 
toms in  such  cases  resemble  those  produced  by  irritant  mineral 
poisons  such  as  arsenic :  nausea  and  vomiting,  vertigo,  dryness 
of  the  mouth,  sense  of  burning  and  constriction  in  the  throat, 
difficulty  in  swallowing,  cramps  and  griping  pain  in  the  bowels, 
constipation  or  diarrhea,  general  prostration  or  even  collapse. 
Vaughan  isolated  from  poisonous  cheese  a  ptomaine  which 
he  called  tyrotoxicon.  It  appears,  however,  that  other  toxins 
may  be  present  in  cheese,  and  that  tyrotoxicon  is  a  somewhat 
rare  poison.  Vaughan  holds  that  bacteria  of  the  colon  group 
play  an  important  part  in  producing  poisons  in  milk  and  cheese. 

To  prevent  the  alteration  by  bacteria  of  milk  intended  for 
the  food  of  infants,  the  practice  of  sterilizing  milk  has  been 
largely  in  vogue.  Unfortunately,  during  sterilization  the  milk 
undergoes  some  kind^  of  alteration  which  makes  it  disagree 
with  certain  infants.  Furthermore,  organisms  possessing 
very  resistant  spores — the  hay  bacillus  and  the  potato  bacillus- 
are  apt  to  be  present  in  milk,  and  these  are  not  killed  by  any 
process  to  which  the  milk  should  be  subjected  for  infant 
feeding.  Least  of  all  does  sterilization  purify  milk  in  which 
bacterial  poison  has  already  formed. 

In  regard  to  the  destruction  of  the  tubercle  bacillus  in  milk 
Glinther*has  this  to  say:  Morgenroth  found  that  10  minutes' 
heating  at  70°  C.  does  not  kill  the  tubercle  bacillus,  and  neither 
does  2  hours  at  55°  C.  The  same  authority  found  that  in  order 
to  destroy  the  tubercle  bacillus  in  milk  from  a  tuberculous  cow 

*Loc.  cit. 


154  MANUAL    OF    BACTERIOLOGY. 

containing  the  tubercle  bacillus  it  is  necessary  to  heat  the  milk 
for  3  hour  at  55°  C.  or  30  minutes  at  70°  C.,  or  from  3  to  5 
minutes  at  100°  C.  Bang  found  that  2  minutes,  heating  at 
00°  C.  caused  a  marked  diminution  in  the  virulence  of  the 
tubercle  bacilli  in  milk.  Morgenroth  found  that  a  very  short 
heating  at  100°  C.  sufficed  to  diminish  the  virulence 
materially. 

Rosenau  *  found  that  in  milk  the  tubercle  bacillus  loses  its 
virulence  and  infective  power,  i.  e.,  is  killed,  by  exposure  to 
60°  C.  for  twenty  minutes. 

The  investigations  of  Park  and  Holt|  show  that  in  New 
York  City  the  number  of  bacteria  in  milk  is  much  smaller  in 
winter  than  in  summer,  and  has  little  effect  on  the  health  of 
infants  during  cold  weather;  but  that  in  warm  weather  with 
milk  of  average  quality  the  infants  who  received  sterilized 
milk  throve  on  the  average  much  better  than  those  who  re- 
ceived raw  milk. 

The  process  called  pasteurization  is  designed,  not  to  sterilize 
the  milk  completely,  but  to  destroy  the  vegetative  forms 
of  bacteria,  and  to  destroy  the  ordinary  pathogenic  bacteria 
with  which  the  milk  might  possibly  be  contaminated.  J  The 
milk  is  subjected  to  a  temperature  of  only  about  70°  to  75°  C. 
This  temperature  is  less  likely  to  produce  alteration  in  the 
milk  than  sterilization  by  steam  at  100°  C.  According  to  Free- 
man, milk  which  had  been  pasteurized  at  75°  C.  and  distrib- 
uted among  the  poor  people  of  New  York  City,  whose  homes 
were  not  supplied  with  ice,  usually  kept  very  well  even  in  the 
summer  time. 

The  number  of  bacteria  in  milk  may  be  reduced  consider- 
ably by  the  use  of  the  centrifuge  (separator). 

*Bull.  No.  42,  Hyg.  Lab.,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.,  Wash. 
pp.  1-85.  Jan.,  1908. 

\ Archives  of  Pediatrics.     December,  1903. 

|Theobald  Smith.  The  Thermal  Death-point  of  Tubercle  Bacilli  in  Milk.  etc. 
Journal  of  Experimental  Medicine.  Vol.  IV.,  p.  217. 


THE    BACTERIA    OF    SOIL,    AIR,    WATER,    ETC.  155 

St.  John  and  Pennington*  conclude  from  their  study  of  the 
effects  of  pasteurization  of  milk,  that  there  is  a  restraining 
power  in  raw  milk  lasting  at  least  to  the  curdling  point  which 
is  destroyed  or  reduced  in  milk  heated  to  79°  C. 

Rosenau,!  from  a  consideration  of  the  various  statements 
made  in  favor  of  and  those  which  are  against  pasteurization, 
comes  to  the  conclusion  that  heating  of  milk  has  certain  dis- 
advantages which  should  be  given  due  consideration,  but  since 
it  is  the  means  of  saving  many  lives  of  children,  particularly  dur- 
ing the  summer  months,  pasteurization  is  to  be  recommended. 
The  ideal  condition  would  be  milk  obtained  so  clean  and 
kept  so  cold  that  there  would  be  no  necessity  for  pasteurization, 
but  such  conditions  are  not  found  in  practice.  Rosenau 
does  not  regard  the  statement  that  pastuerization  "devitalizes" 
the  milk  as  having  any  force  or  indeed  any  exact  meaning. 
He  does  not  regard  the  idea  that  pasteurized  milk  is  a  cause  of 
scurvy  as  well  founded.  Under  conditions  as  they  exist,  the  con- 
sensus of  opinion  among  those  best  qualified  to  pronounce  upon 
the  subject  seems  to  favor  the  pasteurization  of  milk,  at  least 
for  infant  feeding.  Theobald  Smith  J  points  out  that  the  objec- 
tion raised  by  some  to  pasteurization  that  it  conceals  dirt  is 
erroneous  for  the  reason  that  the  bacteria  coming  from  dirt  are 
spore-bearers,  and  are  not  killed  by  pasteurization.  Rosenau 
further  states  that  comparative  observations  have  shown  that 
children  thrive  quite  as  well  on  pasteurized  as  on  raw  milk, 
that  pasteurized  milk  is  in  fact  more  easily  digested  than  raw 
milk.  But  on  the  other  hand,  there  are  many  who  object  to 
pasteurization  of  milk,  and  the  question  can  hardly  yet  be 


*St.  John  and  Pennington.     Journal  oj  Infectious  Diseases.     IV.,  No.  4, 1907, 

P-  655- 

fRosenau.  Milk  and  its  Relation  to  the  Public  Health.  Pasteurization 
Bulletin  41.  Hyg.  Lab.,  U.  S.  Pub.  Health  and  Mar.  Hosp.  Serv.  Wash.,  D.  C., 
pp.  589—624. 

^Theobald  Smith.  Am.  Journ.  Pub.  Health  and  Journ.  Mass.  Assn.  Bds. 
Health.  Vol.  17,  1907,  p.  200.  Quoted  by  Rosenau.,  loc  cit. 


156  MANUAL   OF    BACTERIOLOGY. 

regarded  as  settled.  Nevertheless,  the  advantages  seem 
certainly  to  greatly  outweigh  the  disadvantages. 

In  regard  to  the  pasteurization  of  milk,  Rogers*  has  this  to 
say: 

"Examination  of  milk  by  many  bacteriologists  shows  that 
the  milk  used  in  American  cities  is  usually  badly  contaminated 
by  bacteria. 

"Increased  public  interest  in  the  milk-supply  has  resulted 
in  more  rigid  municipal  regulations  and  inspection,  but  prog- 
ress is  necessarily  slow  and  pasteurization  is  frequently  re- 
sorted to  in  order  to  increase  the  length  of  time  that  the  milk 
will  remain  sweet  and  to  reduce  the  danger  from  spread  of 
infectious  diseases. 

"The  objection  is  frequently  made  that  pasteurization,  by 
destroying  the  lactic-acid  bacteria,  allows  the  development  of 
other  less  desirable  bacteria  which,  without  affecting  the  taste 
of  the  milk,  make  it  actually  dangerous,  especially  as  a  food 
for  young  children. 

"It  is  well  established,  however,  that  under  certain  circum- 
stances the  intestinal  troubles  of  children  may  be  reduced  by 
pasteurization  of  milk. 

"Milk  was  pasteurized  .under  laboratory  conditions  in  a 
continuous  machine  at  85°  C.  (185°  F.),  the  bacteria  being 
reduced  from  over  10,000,000  per  cubic  centimeter  to  less 
than  500  per  cubic  centimeter. 

"Milk  held  at  20°  C.  (68°  F.).— In  the  unheated  milk  the 
lactic  bacteria  increased  rapidly  and  the  milk  became  acid  in 
about  12  hours.  The  peptonizing  bacteria  increased  in  6 
hours  to  about  5,000,000  per  cubic  centimeter  and  then  de- 
creased slowly. 

"In  the  heated  milk  the  peptonizing  bacteria  increased 
rapidly  after  12  hours,  and  the  milk  was  usually  curdled  in  48 
hours,  with  a  disagreeable  taste  and  odor.  Occasionally 

*Bureau  of  Animal  Industry.     Bui.  No.  73,  1905 


THE    BACTERIA    OF   SOIL,    AIR,    WATER,    ETC.  157 

lactic  bacteria  survived  pasteurization  and  multiplied  rapidly 
after  24  hours,  completely  inhibiting  the  peptonizing  bacteria. 

"Milk  held  at  10°  C.  (50°  F.). — In  unheated  milk  the  growth 
of  bacteria  and  the  consequent  curdling  of  the  milk  was  much 
retarded.  The  average  milk  did  not  contain  sufficient  acid  to 
affect  the  taste  until  it  was  over  48  hours  old.  The  propor- 
tion of  peptonizing  to  lactic  bacteria  was  greater  than  at  the 
higher  temperature  and  the  taste  of  the  milk  occasionally 
showed  the  influence  of  the  former. 

"In  the  pasteurized  milk  the  bacteria  increased  very  slowly, 
and  in  nearly  every  case  the  milk  was  unchanged  in  taste  and 
appearance  96  hours  after  pasteurization.  In  only  two  of 
fourteen  cases  was  there  a  marked  increase  of  peptonizing 
bacteria.  The  predominating  bacteria  were  species  having 
little  or  no  effect  on  milk. 

"The  lactic  bacteria  inhibited  the  development  of  the 
peptonizing  bacteria  only  when  they  had  developed  sufficient 
acid  to  render  the  milk  unfit  for  use. 

"It  seems  probable  that  the  acid  had  a  distinct  inhibitory 
action  on  the  proteolytic  enzymes  of  the  peptonizing  bacteria. 

"If  milk  could  be  pasteurized  commerically  in  such  a  way 
that  the  bacteria  would  be  reduced  to  a  few  hundred  per  cubic 
centimeter  and  held  at  a  low  temperature  until  used,  it  would 
be  perfectly  safe  for  48  hours  or  even  72  hours.  Under  these 
circumstances  it  would  probably  be  in  better  condition  after 
this  long  period  than  ordinary  city  milk  at  the  time  it  is  delivered. 
How  closely  these  conditions  could  be  approximated  commer- 
cially is  another  question." 

Since  the  publication  of  the  above  bulletin,  it  -has  been 
demonstrated  in  some  dairy  plants  that  such  conditions  can 
be  brought  about  upon  a  commercially  profitable  scale,  and 
it  would  seem  justifiable  on  the  part  of  health  authorities  to 
demand  that  milk  dealers  comply,  or  that  the  municipal 
authorities  should  establish  pasteurizing  plants. 


158  MANUAL    OF    BACTERIOLOGY. 

The  great  interest  which  is  taken  by  the  public  and  by  physi- 
cians in  the  subject  of  pure  milk  has  led  some  dairyman  to 
take  steps  to  prevent  contamination  of  the  milk  by  cleanliness  in 
the  barnyard  and  stable,  and  in  careful  cleansing  of  the  udder? 
of  the  cows,  the  hands  of  the  milkers  and  the  milk-cans.  The 
dairy-rooms  are  also  kept  scrupulously  clean  in  the  better 
class  of  dairies,  and  the  floors  are  kept  wet  to  avoid  dust* 
The  use  of  ice  in  cooling  the  milk  as  soon  as  it  is  drawn,  and 
in  transportation  is  also  used,  and  this  serves  to  prevent  the 
multiplication  of  bacteria.  In  many  dairies  the  cattle  from 
which  the  milk  is  obtained  are  regularly  inspected  at  intervals 
by  veterinary  surgeons  as  well  as  subjected  periodically  to  the 
tuberculin  test.  The  surroundings  and  drainage  of  the  stables 
are  investigated  by  physciians  and  sanitary  engineers.  The 
milk  is  also  regularly  analyzed  by  a  chemist  and  bacteriologist. 
It  has  been  found  possible  by  such  precautions  to  reduce  the 
number  of  bacteria  in  milk  to  a  few  thousands  per  cubic 
centimeter  or  even  much  less. 

Other  articles  of  food  which  are  eaten  after  little  or  no  cook- 
ing such  as  salads,  green  vegetables,  fruits,  and  the  like,  may 
become,  under  exceptional  circumstances,  agents  for  conveying 
infectious  diseases.  Conn  showed  that  there  was  good  reason 
for  attributing  an  epidemic  of  typhoid  fever  among  students 
at  Middletown,  Connecticut,  to  raw  oysters.  After  having 
been  collected  from  the  oyster-beds,  these  oysters  were  placed 
in  a  small  stream  to  fatten,  where  they  were  exposed  to  con- 
tamination from  a  sewer.  Into  this  sewer  the  discharges  of  a 
case  of  typhoid  fever  were  found  to  have  been  running  at  the 
time  when  the  oysters  were  fattening.  An  epidemic  at  Atlantic 
City,  New  Jersey,  in  1902,  was  traced  to  nearly  similar  causes 
and  conditions.! 


*W.  H.  Park.     Journal  o}  Hygiene.     Vol.  I.,  1901. 
^Philadelphia  Medical  Journal.     November  i,  1902. 


i 


THE    BACTERIA   OF    SOIL,    AIR,    WATER,    ETC.  159 

The  ordinary  processes  for  curing  and  salting  meat  cannot 
be  relied  upon  to  destroy  pathogenic  bacteria. 

Cases  of  botulism,  or  poisoning  by  eating  oysters,  fish,  meat 
in  the  form  of  sausage  or  canned  meat,  and  other  articles  of 
food,  are  not  rare.  They  are  due  to  products  of  bacterial  de- 
composition, as  in  the  case  of  those  poisoned  by  milk  and 
cheese  already  mentioned.  Such  affections  are  quite  com- 
monly called  ptomaine  poisoning.  A  number  of  bacteria  exist 
which  are  capable  of  affecting  injurious  changes  in  meat  and 
other  foods  either  before  or  after  ingestion.  Among  these 
are  an  anaerobic  bacillus  described  by  Van  Ermengem  (B. 
botulinus),  various  members  of  the  groups  represented  by  B. 
proteus  and  B.  coli  communis  (including  paracolon  bacilli), 
and  the  bacillus  enteritidis  of  Gaertner.  In  the  case  of  B. 
enteritidis  a  genuine  infection  of  the  patient  and  gastroenteritis 


*See  Vaughan  and  Novy.  The  Cellular  Toxins.  1902.  Ohlmacher.  Food 
Intoxication  from  Oatmeal.  Journal  of  Medical  Research.  Vol.  VII.,  p.  420. 
Galeotti  and  Zardo.  Centralblatt  fur  Bakteriologie.  Vol.  XXXI.  1902.  Orig. 
P-  593- 


CHAPTER  IV. 
THE  BACTERIA  OF  THE  NORMAL  HUMAN  BODY. 

ALTHOUGH  there  is  considerable  discrepancy  in  the  results  of 
various  investigations  in  regard  to  the  matter,  it  would  appear 
that  the  solid  tissues  of  the  animal  body,  the  blood  and  lymph, 
and  the  cavities  that  have  no  connection  with  the  outer  world, 
are  often  free  from  bacteria.*  So  also  the  maxillary,  eth- 
moidal  and  frontal  sinuses,  middle  ear,f  urinary  bladder, 
uterus  and  Fallopian  tubes,  and  to  a  less  extent  the  lungs  t  and 
gall-bladder, §  although  having  external  connections,  are 
usually  sterile  when  in  a  healthy  condition.  When  bacteria  do 
enter  the  tissues  from  any  of  the  surfaces  their  progress  is 
checked  by  means  of  the  activities  of  the  cells  or  fluids  of  the 
body,  and  if  they  succeed  in  penetrating  to  any  considerable 
distance  their  advance  is  usually  arrested  by  the  nearest  group 
of  lymph-nodes,  which  appear  to  be  important  safeguards  for 
preventing  the  dissemination  of  bacteria  throughout  the  body. 
As  a  rule,  the  sections  of  the  mucous  membranes  are  inimical 
to  bacteria. 

The  skin,  1 1  as  might  be  expected,  is  liable  to  have  upon  it 

*Ford  found  small  numbers  of  bacteria  in  the  normal  organs  of  rabbits,  cats 
and  dogs  in  the  majority  of  those  examined.  The  species  of  bacteria  obtained 
were  mostly  common  saprophytes,  and  to  some  extent  constant  in  the  same 
kind  of  animal.  Journal  of  Hygiene.  Vol.  I.  1901. 

tCalamida  and  Bertarelli.  Centralblatt  }ilr  Bakteriologie.  Vol.  XXXII.  1902. 
Orig.  p.  428.  Torne.  Ibidem.  XXXIII.  1903.  p.  250.  Hasslauer. 
Ibidem.  Referate.  XXXII,  p.  174.  An  examination  of  these  articles  will 
show  that  investigators  disagree  somewhat,  with  regard  to  the  sterility  of  these 
cavities. 

tSee  Wadsworth.     American  Journal  Medical  Sciences.     May,  1904. 

§See  Bacteriology  of  the  Gall-bladder  and  its  JDucts.  American  Journal 
Medical  Sciences.  Vol.  CXXIIL,  p.  372. 

|[Sabouraud.     La  Peau  Humaine,  etc.     Bulletin  de  V  Institut  Pasteur.     II 
1904.     Pages  233,  282. 

1 60 


THE   BACTERIA   OF  THE    NORMAL   HUMAN   BODY.         l6l 

numerous  bacteria,  especially  micrococci,  and  moulds.  The 
staphylococcus  pyogenes  aureus,  the  streptococcus  pyogenes, 
the  bacillus  pyocyaneus  and  the  bacillus  coli  communis  some- 
times occur  on  the  skin.  According  to  Welch,  it  always  con- 
tains the  staphylococcus  epidermidis  albus,  which  may  be  a 
form  of  the  staphylococcus  pyogenes  albus.  This  organism  is 
of  some  importance  to  surgeons  on  account  of  its  relation  to 
the  cleansing  of  the  skin  before  operations.  It  seems  impos- 
sible, by  any  amount  of  cleaning,  to  dislodge  all  of  the  germs  in 
the  skin  especially  those  under  the  nails. 

The  bacteria  of  the  exposed  mucous ~  membranes  like  the 
conjunctiva*  and  the  nasal  cavityf  and  the  mouth  cavity 
naturally  fluctuate  both  in  quantity  and  quality;  they  consist, 
in  fact,  of  those  which  happen  to  fall  upon  the  surface  or  are 
drawn  in  from  the  external  air. 

In  the  mouth,  however,  there  is  a  certain  group  of  organisms 
more  or  less  characteristic  of  it,  many  of  which  have  not  been 
successfully  cultivated.  These  have  been  thoroughly  studied 
by  Miller,  to  whose  works  students  are  referred.  J 

Several  species  of  spirilla  have  been  discovered  in  the  mouth 
and  are  found  along  the  margins  of  the  gums.  The  leptothrix 
buccalis,  and  related  organisms  which  have  a  long,  ribbon-like 
form,  also  occur  in  the  mouth.  The  micrococcus  lanceolatus 
(or  pneumococcus)  is  present  in  many  human  mouths.  In 
15  to  20  per  cent,  of  human  mouths  this  organism  is  sufficiently 
virulent  to  produce  fatal  septicemia  when  inoculated  into 
susceptible  animals.  Pyogenic  bacteria,  especially  streptococci 
occur  frequently,  although  not  regularly,  in  the  mouth.  Strep- 
tococci very  commonly  occur  on  the  tonsils.  Putrefactive 

*Randolph,  Pusey,  Gifford.  Journal  American  Medical  Association.  Oct. 
3,  1903. 

fHasslauer.  Die  Bakterienflora  der  gesunden  und  kranken  Nasenschleim- 
haut.  Centralblatt  fur  Bakteriologie.  Vol.  XXXIII.  1902.  Orig.  p.  47- 

t  Miller.  Microorganisms  of  the  Mouth.  For  a  review  on  the  bacteria  of 
the  mouth,  see  Madzar.  Centralblatt  far  Bakteriologie.  Vol.  XXXI.  Ref. 
p.  489.  Vol.  XXXII.  p.  609. 


1 62  MANUAL    OF    BACTERIOLOGY. 

bacteria  acting  on  particles  of  food  about  the  teeth  produce  the 
bad  odor  from  the  mouths  of  persons  of  careless  habits.  Ac- 
cording to  Miller,  bacteria  play  an  important  part  in  the  pro- 
duction of  dental  caries.  Certain  of  the  bacteria  of  the  mouth 
produce  fermentation  in  the  vicinity  of  the  teeth  with  the 
formation  of  acids,  which  dissolve  the  calcium  'salts  of  the 
teeth.  The  softening  and  destruction  of  the  decalcified 
matrix  is  then  accomplished  by  other  forms. 

The  expired  air  coming  from  the  mouth  and  nose,  con- 
trary to  the  popular  notion,  is  free  from  bacteria,  excepting 
those  which  become  forcibly  detached,  as  by  efforts. of  sneezing 
and  coughing. 

McKee*  found  that  in  the  great  majority  of  normal  con- 
junctivas the  ordinary  pyogenic  bacteria  and  the  bacillus 
xerosis  are  present.  He  quotes  Eyref  as  saying  that  the  con- 
junctival  sac  frequently  contains  bacteria  which  may  or  may 
not  be  pathogenic;  but,  on  the  other  hand  that  it  may  be 
sterile,  due  to  the  mechanical  flushing  of  the  mucous  surface 
by  the  lachrymal  secretion,  aided  perhaps  by  the  bactericidal 
property  of  the  latter. 

Among  the  other  exposed  mucous  "surfaces,  the  urinary 
meatus  and  the  vagina  may  be  included.  The  urinary  meatus 
and  at  least  part  of  the  urethra  will  be  found  to  contain  bacteria, 
which,  in  health,  should  be  non-pathogenic,  although  interest 
attaches  to  the  fact  that  diplococci  have  been  described  which 
behaved  with  stains  in  the  same  manner  as  the  gonococcus 
(pseudogonococci) . 

There  has  been  much  dispute  as  to  whether  or  not  the 
pyogenic  bacteria  occur  in  the  vagina  normally.  But  there 
appears  abundant  evidence  going  to  show  that  while  the  vagina 
may  be  free  from  pathogenic  bacteria  it  often  if  not  usually 
harbors  pyogenic  bacteria.  While  it  is  true  that  the  normal 

*Reprint  from  Montreal  Med.  Journ.     Jan.,  1906. 

^Lancet.     Dec.  21.  1895.     Journ.  Pathol.  and  Bacter.     July,  1896. 


THE    BACTERIA    OF    THE    NORMAL    HUMAN    BODY.          163 

secretion  of  the  vagina  has  a  bactericidal  influence  which  may 
be  attributed  in  part  to  its  acidity,  this  does  not  seem  to  be  ef- 
fective  at  all  times.  The  upper  part  of  the  normal  cervix  uteri 
is  sterile,  while  bacteria  are  present  in  the  lower  part. 

McDonald*  quotes  from  Bumm  and  Sigwart  to  show  that  streptococcus  is 
present  in  the  secretions  from  the  uterus  in  38  per  cent,  of  cases  in  women  during 
the  latter  stages  of  pregnancy,  and  that  they  further  more  state  the  belief  that 
this  organism  is  present  in  75  per  cent,  in  such  cases.  That  of  the  cases  showing 
the  presence  of  streptococci,  24  per  cent,  had  fever.  She  agrees  with  the  authors 
cited  that  the  presence  of  streptococcus  is  not  sufficient  grounds  for  a  diagnosis  of 
puerperal  infection.  The  futility  and  harmfulness  of  curettment  in  cases  of 
general  infection  is  evident. 

According  to  Doderlein,  the  properties  of  the  vaginal  secretion  are  due  to 
bacilli  which  very  commonly  occur  in  it.  The  secretion  is  most  abundant  and 
important  during  pregnancy,  f 

The  smegma  of  the  external  genitals  contains  numerous 
bacteria,  among  which  are  frequently  found  bacilli  which  re- 
tain their  color  after  treatment  with  acids  in  the  Gabbett 
method  for  staining  tubercle  bacilli.  It  is  uncertain  whether 
these  bacilli  form  a  special  group  of  organisms  by  themselves, 
having  as  one  of  their  properties  the  power  of  retaining  the  stain 
after  acids,  or  whether  they  are  bacilli  of  no  particular  sort, 
which  resist  acids  after  staining  owing  to  the  oily  material  with 
which  they  have  been  impregnated  in  this  peculiar  secretion. 
These  organisms  must  be  taken  into  account  in  examining 
urine  or  other  secretions  for  tubercle  bacilli,  for  particles  of 
smegma  might  be  accidentally  present.  Usually  the  employ- 
ment of  alcohol  after  the  acid  will  remove  the  color  from  the 
smegma  bacilli  (Hueppe).  Sometimes  smegma  bacilli  are  as 
resistant  as  tubercle  bacilli  to  decolorizing  agents  (Welch)  (see 
page  34).  Similar  acid-proof  bacilli  occur  about  the  genitals 
of  the  domestic  animals.! 

*Reprint  from  A merican  M edicine.  Vol.  XI.,  No.  7.  Feb.  17,  1906,  pp.  231- 
238. 

tj.  W.  Williams.  Obstetrics.  A  Text-book,  etc.  1908.  Wadsworth. 
American  Journal  of  Obstetrics.  Vol.  XLIII.  1901. 

|Cowie.     Journal  Experimental  Medicine.     Vol.' V.,  p.  205. 


164  MANUAL    OF    BACTERIOLOGY. 

Bacteria  are  always  present  to  a  greater  or  less  extent  in  the 
stomach  and  intestines  except  for  a  few  hours  after  birth. 
The  alimentary  tract  of  new-born  infants  and  the  meconium 
are  sterile.  In  from  four  to  eighteen  hours  organisms  begin  to 
appear.  They  may  enter  either  from  the  mouth  or  the  anus. 
There  seems  to  be  no  constancy  in  the  nature  of  the  forms 
which  are  found  at  first,  but  their  character  depends  upon  the 
surroundings. 

The  species  of  bacteria  found  in  the  stomach  are  less  con- 
stant than  those  of  the  intestines;  and  under  normal  circum- 
stances they  seem  to  be  those  introduced  from  the  mouth. 
Different  investigators,  at  all  events,  have  met  with  quite 
different  species  in  the  stomach.  It  appears  that  the  hydro- 
chloric acid  (about  2  parts  per  thousand)  present  in  the  gastric 
juice  at  the  height  of  digestion  possesses  decided  germicidal 
properties.  This  germicidal  power  exercises  a  restraining  in- 
fluence upon  fermentation  due  to  bacteria,  and  probably  serves 
as  a  safeguard  against  the  introduction  of  pathogenic  germs 
into  the  intestines.  That  is  particularly  important  in  the  case 
of  the  spirillum  of  cholera,  which  is  excessively  sensitive  to  the 
action  of  acids.  Nevertheless,  many  bacteria  are  able  to  reach 
the  intestines  uninjured,  as  the  acidity  of  the  gastric  juice  does 
not  reach  its  height  until  some  hours  after  eating.  Such 
bacteria  will  be  those  which  are  most  resistant  and  those  which 
form  spores.  In  the  intervals  when  hydrochloric  acid  is  ab- 
sent from  the  stomach,  lactic  acid  appears.  It  is  formed  from 
carbohydrates  by  a  large  number  of  species  of  bacteria.  In 
conditions  of  fermentation,  sacrina  ventriculi  and  yeasts  may 
be  present  in  large  numbers;  in  the  healthy  stomach  they  occur 
in  much  smaller  numbers. 

The  intestine  of  the  infant  in  whom  feeding  has  become 
well  established  was  found  by.Escherich  to  contain  two  prin- 
cipal species  of  bacteria — in  the  lower  part  of  the  intestine  the 
bacillus  coli  communis,  in  the  upper  part  the  bacilus  lactis  ; 


THE    BACTERIA    OF    THE    NORMAL    HUMAN    BODY.          165 

aerogenes.  More  recently  it  has  been  shown  that  the  stools 
of  milk-fed  infants,  and  to  a  less  extent  of  adults,  contain  large 
numbers  of  anaerobic  bacilli,  which  stain  by  Gram's  method 
(bacillus  bifidus — Tissier,  bacillus  acidophilus — Moro) .  These 
bacteria  have  not  been  fully  studied*. 

The  number  of  bacteria  in  a  milligram  of  human  fecal 
matter  has  been  estimated  at  from  seventy  thousand  to  thirty- 
three  million. f  It  is  estimated  that  about  one-third  of  the 
fecal  matter  of  adults  if  dried  would  be  found  to  consist  of 
bacteria.  J  The  small  intestine  of  adults  has  been  found  by 
different  observers  to  contain  very  different  species.  §  The 
majority  of  these  appear  to  have  been  introduced  from  the 
mouth  in  food  or  water.  The  bacillus  coli  communis,  however, 
occurs  invariably  in  health  not  only  in  the  intestine  of  man,  but 
also  in  that  of  many  animals,  especially  in  the  lower  part.  || 
The  pyogenic  micro  cocci  yery  often  occur  in  the  intestine. 

In  the  case  of  ruminant  animals  like  the  cow  and  sheep, 
the  decomposition  of  cellulose,  which  forms  so  large  a  part  of 
their  food,  appears  to  be  affected  by  bacteria.  Bacteria  having 
this  power  are  constantly  found  in  the  stomachs  of  ruminants. 
The  best  known  species  is  that  called  bacillus  amylobacter.  It 
is  questionable  whether  the  products  of  the  decomposition  of 
cellulose  have  any  nutritive  value. 

Pasteur  some  years  ago  expressed  the  opinion  that  if  animals  could  be  placed 
in  such  surroundings  that  bacteria  could  be  excluded  from  the  alimentary  canal 
and  the  food,  life  would  be  impossible.  This  view  has  excited  much  controversy, 
and  was  apparently  disproved  by  the  experiments  of  Nuttall  and  Thierfelder. 


*Metchnikoff.  Les  Microbes  Intestinaux.  Bulletin  de  VInstitut  Pasteur. 
May  15  and  30,  1903. 

t  Our  method  for  estimating  the  number  of  bacteria  in  feces  see  Steele. 
Journ.  Am.  Med.  Assn.,  XLIX.,  Aug.  24,  1907,  p.  647. 

%Strassburger  Centrallbel.  f.  Bakt.,  etc.     Ref.  Bd.  32.  p.  561. 

§Ford.  Classification  of  Intestinal  Bacteria,  etc.  Studies  from  the  Royal 
Victoria,  Montreal.  March,  1903. 

||Moore  and  Wright.  Bacillus  coli  communis  from  Certain  Species  of  Domes- 
ticated Animals.  American  Medicine.  March,  1902. 


1 66  MANUAL    OF    BACTERIOLOGY. 

These  investigators  succeeded  in  removing  guinea-pigs  from  the  mother  by 
Cesarean  section,  and  in  keeping  them  alive  in  sterile  surroundings,  upon  sterile 
food,  so  that  the  contents  of  the  alimentary  canal  remained  sterile.  Schottelius, 
who  worked  with  chickens,  obtained  contrary  results,  however;  so  that  this 
interesting  question  is  still  undecided 


CHAPTER  V. 
BACTERIA  IN  DISEASE. 

To  THE  physician  and  the  student  of  medicine  the  study  of 
bacteriology  is  interesting  chiefly  on  account  of  the  great  im- 
portance attributed  to  bacteria  in  producing  disease.  The 
presence  in  an  organism  of  one  or  a  number  of  organisms  of 
another  species,  which  flourish  as  parasites  upon  the  first,  is  a 
phenomenon  of  very  wide  occurrence  in  nature.  It  is,  in  fact, 
nearly  universal.  It  may  be  observed  among  plants  as  well  as 
animals,  for  example  in  the  familiar  galls  seen  on  some  of  the 
higher  plants,  and  mostly  caused  by  the  larvae  of  insects  har- 
bored by  the  plant.  We  also  find  animals,  such  as  tapeworms 
and  the  trichina  spiralis,  living  as  parasites  upon  other  animals. 
The  conditions  favorable  to  the  growth  of  certain  bacteria 
make  them  peculiarly  suited  to  leading  a  parastic  existence. 
The  fact  that  they  possess  no  chlorophyll,  and  that  they  are 
therefore  unable  to  form  carbon  compounds  from  the  carbon 
dioxide  of  the  atmosphere,  renders  it  necessary  for  them  to 
secure  such  compounds  from  pre-existing  organic  matter. 
Most  of  them,  furthermore,  flourish  better  when  they  are  able 
to  obtain  nitrogenous  food  from  organic  matter  rather  than 
from  inorganic  salts  containing  nitrogen.  Most  bacteria,  those 
known  as  saprophytes,  find  the  necessary  nutriment  in  the 
dead  bodies  of  other  animals  and  plants;  but  some  of  them, 
those  known  as  parasites,  flourish  upon  the  living  bodies  of 
other  plants  and  animals  and  produce  disease. 

The  phenomena  of  disease,  as  has  been  well  established,  are 
due  in  a  number  of  cases  to  the  numerous  waste  products  of  the 
activities  of  bacteria,  which  act  as  poisons  to  the  host. 


1 68  MANUAL    OF    BACTERIOLOGY. 

The  diseases  of  plants  known  to  be  caused  by  bacteria  are 
not  very  numerous.  Among  them  may  be  mentioned  pear- 
blight,  due  to  micrococcus  amylovorus.*  Among  lower 
animals  bacteria  very  frequently  produce  diseases — for  example, 
chicken-cholera,  symptomatic  anthrax  in  cattle,  erysipelas  of 
swine,  tuberculosis,  anthrax  and  glanders  in  various  animals, 
"red  leg"  in  frogs. t 

These  are  some  of  the  diseases  in  which  bacteria  have  been 
shown  definitely  to  be  the  cause.  It  is  not  enough  in  any  case 
merely  to  find  bacteria  to  establish  the  connection  between 
them  and  the  disease.  Koch's  postulates,  as  they  are  called, 
given  below,  must  be  complied  with  in  order  to  prove  that  any 
microorganism  is  the  cause  of  a  particular  disease: 

First.  That  the  organism  should  always  be  found  micro- 
scopically in  the  bodies  of  animals  having  the  disease;  that 
it  should  be  found  in  that  disease  and  no  other;  that  it  should 
occur  in  such  numbers  and  be  distributed  in  such  a  manner  as 
to  explain  the  lesions  of  the  disease. 

Second.  That  the  organism  should  be  obtained  from  the 
diseased  animal  and  propagated  in  pure  culture  outside  of 
the  body. 

Third.  That  the  inoculation  of  these  germs  in  pure  cultures, 
which  had  been  freed  by  successive  transplantations  from  the 
smallest  particle  of  matter  taken  from  the  original  animal, 
should  produce  the  same  disease  in  a  susceptible  animal. 

Fourth.  That  the  organism  should  be  found  in  the  lesions 
thus  produced  in  the  animal. 

A  moment's  consideration  will  show  that  it  is  impossible  to 
comply  with  all  these  postulates  in  the  investigation  of  all  in- 
fectious diseases;  for  in  some  cases  the  organisms  causing  the 
diseases  have  not  yet  been  observed,  and  yet  there  is  abundant 
proof  that  they  exist  in  certain  tissues  of  animals  suffering  from 

*See  E.  Smith.  Centralblatt  fur  Bakteriologie,  etc.  Zweite  Abtheilung.  Bd. 
V.,  p.  271  ;Bd.  VIL,  p.  88. 

t  Norris  and  Emerson.     Journal  of  Experimental  Medicine,     Vol.  VII.,  p  30. 

•: 


BACTERIA    IN    DISEASE.  169 

j  these  diseases.     Thus,  it  is  well  known  that  the  hydrophobia 
I  virus  is  to  be  found  constantly  in  the  brain  and  cord  of  animals 
f  suffering  from  rabies,  and  yet  it  has  been  impossible  to  isolate 
and  cultivate  the  organism.     Again,  the  leprosy  bacillus   is 
I  found  always  present  in  the  lesions  of  the  disease,  and  yet  it 
,  has  not  yet  been  cultivated  and  reinoculated.     The  spirocheta 
I  pallida  of  syphilis  has  been  successfully  inoculated  from  syphi- 
litic lesions  into  monkeys,  but  it  has  not  yet  been  cultivated 
!  outside  the  animal  body. 

Still,  although  the  etiology  of  some  diseases  seems  firmly 

established  even  where  all  of  these  postulates  have  not  been 

I  fulfilled,  it  is  nevertheless  the  aim  in  all  cases  to  comply  with 

!  them  as  fully  as  the  nature   of  the  case,  and  the  limits  of 

i  present  knowledge  and  technique  will  permit. 

If  the  fact  is  once  established  in  any  given  case  that  a  dis- 

i  ease  may  be  communicated  from  a  sick  individual  to  a  healthy 

one  it   is  classed  as  an  infectious  disease,  and  infectious  dis- 

f  eases  are  all  caused  by  some  living  parasite.     Consequently: 

An  infectious  disease  is  a  disease  which  is  caused  by  a  micro- 

|  organism  growing  in  the  body  of  the  animal  having  the  disease. 

i  Such  microorganisms  are  usually  bacteria,  but  not  always;  for 

I  example,  malaria  is  produced  by  a  minute  animal  organism. 

A  contagious  disease  is  one  which  is  acquired  from  direct  or 

indirect  contact  with  an  individual  having  the  disease.     Most 

contagious  diseases  are  infectious,  but  infectious  diseases  are 

not  necessarily  contagious.     The  words  are  often  used  very 

loosely,  and  it  is  no  longer  possible  or  very  desirable  to  draw 

the  line  sharply  between  them.     Fomites  are  the  materials  on 

which  the  infectious  material  is  conveyed. 

A  miasmatic  disease  is  a  variety  of  infection  in  which  the 
microorganisms  are  not  received  from  another  case  of  the  dis- 
ease, but  are  supposed  to  have  been  derived  from  the  external 
world,  particularly  through  foul  air.  This  word  is  less  used 
than  formerly. 


i  yo 


MANUAL    OF    BACTERIOLOGY. 


The  following  is  a  list  of  the  most  important  diseases  of  man 
caused  by  bacteria.  The  proof  as  required  by  the  rules  of 
Koch  is  not  complete  for  all  of  them: 


Tuberculosis, 
Leprosy, 
Glanders, 
Anthrax, 
Tetanus, 

Malignant  edema, 
Bubonic  plague, 
Malta  fever, 
Erysipelas, 


Suppuration  and 
certain  inflamma- 
tory conditions  al- 
lied to  it, 

Epidemic  cerebro- 
spinal  meningitis, 

Gonorrhea, 

Chancroid  or  soft 
chancre, 

Lobar  pneumonia, 


Influenza, 
Diphtheria, 
Typhoid  fever, 
Dysentery    (not 
amebic   dysen- 
tery), 

Asiatic  cholera, 
Relapsing  fever, 
Actinomycosis. 
Syphilis  (probably) . 


Malaria  and  amebic  dysentery  are  caused  by  microscopic 
unicellular  animal  organisms  (protozoa).  It  has  been  claimed 
that  small-pox  is  caused  by  protozoa;  this  view  has  acquired 
added  interest  from  the  recent  researches  of  Councilman. 
Recent  work  indicates  that  the  "sleeping  sickness"  (of  Africa) 
and  some  other  diseases  of  tropical  climates  are  caused  by 
protozoa  (see  appendix). 

Thrush  and  certain  parasitic  skin  diseases  are  caused  by 
fungi  of  more  highly  organized  structure  than  bacteria. 

In  each  of  the  following  diseases  there  is  good  reason  to 
think  that  the  cause  is  some  kind  of  microorganism,  but  it  has 
not  yet  been  discovered : 

Chicken-pox,  measles,  scarlet  fever,  German  measles,  mumps, 
whooping-cough,*  yellow  fever,  typhus  fever,  rabies,  dengue. 

Rheumatic  fever  and  beri-beri  would  be  placed  in  this  list 

by  many  writers. 

• 

*Bordet  and  Genou*  have  isolated  an  organism  in  whooping-cough  from  spu- 
tum by  the  use  of  a  mixture  of  rabbit's  blood  with  agar  and  a  small  amount  of 
glycerin  extract  of  potato.  They  state  that  another  organism  resembling  the 
influenza  bacillus  often  presents  a  serious  obstacle  to  the  isolation  of  the  real 
whooping-cough  bacillus.  Ann.  del' lust.  Past.  XXI.,  No.  9,  Sept.  25,  1907. 
PP-  727-732. 


BACTERIA    IN    DISEASE.  171 

The  causes  of  these  diseases  have  been  very  carefully  sought 
for  by  ordinary  bacteriological  methods  with  indecisive  results. 
Some  of  them  no  doubt  are  due  to  bacteria.  In  recent  years 
numerous  observers  have  described  a  diplococcus  or  short 
streptococcus  as  the  cause  of  rheumatic  fever  or  acute  rheu- 
matism. In  the  case  of  yellow  -fever  Sanarelli  described  an 
organism  (bacillus  icteroides)  as  its  cause,  but  his  view  is  not 
upheld  by  most  of  those  who  have  worked  on  yellow  fever.* 
The  bacillus  described  by  a  number  of  observers  as  having 
been  found  in  cases  of  whooping-cough  may  also  be  the  cause 
of  that  disease. f  Bacilli  have  also  been  described  in  cases  of 
measles  on  several  occasions.  The  organism  obtained  by 
Lustgarten  and  that  obtained  by  Joseph  and  Piorkowsky  in  the 
lesions  of  syphilis  are  no  longer  regarded  as  the  cause  of  the 
disease,  but  the  spirocheta  pallida  of  Schaudinn  and  Hoff- 
mann is  probably  the  cause. 

It  is  likely  that  for  some  of  the  diseases  mentioned  other  pro- 
cedures than  the  usual  methods  of  research  will  have  to  be 
devised  in  order  that  the  cause  may  be  discovered.  The  proto- 
zoa may  play  a  part  in  the  etiology  of  some  of  them.  Roux  has 
produced  evidence  to  show  that  contagious  pleuropneumonia 
of  cattle  is  due  to  a  microbe  so  minute  that  it  is  barely  visible 
with  the  highest  powers  of  the  microscope,  so  that  its  outlines 
and  its  morphology  cannot  be  studied.  The  virus  of  this 
disease  remains  virulent  after  being  passed  through  a  Pasteur 
filter,  showing  that  it  is  small  enough  to  go  through  its  pores. 
Similar  experiments  have  succeeded  with  a  number  of  other 
affections  of  animals;  for  example,  foot-and-mouth  disease 
and  hog  cholera  or,  at  any  rate,  an  infectious  epidemic  disease 
presenting  all  the  features  of  hog  cholera.  The  virus  may  pass 
thorugh  a  Pasteur  or  Berkefeld  filter  of  a  certain  coarseness,  but 

*Sanarelli.  La  Semaine  Medicate.  April,  4,  1900.  Reed  and  Carroll. 
Journal  Experimental  Medicine.  Vol.  V. 

fSee  Czaplewski.  Centralblatt  }iir  Bakteriologie.  Bd.  XXIV.  1898.  p. 
865. 


172  MANUAL    OF    BACTERIOLOGY. 

may  be  restrained  by  one  sufficiently  fine.  The  virus  of  rabies  is 
also  probably  filterable.  Reed  and  Carroll  found  that  the 
infective  agent  of  yellow  fever  is  in  the  blood,  and  that 
the  serum  could  produce  yellow  fever  in  a  non-immune  person 
after  filtration  through  a  Berkefeld  filter.*  These  facts  sug- 
gest the  possibility  that  failure  to  find  the  causes  of  some  other 
diseases  may  lie  in  the  fact  that  their  organisms  are  so  small  as 
to  be  nearly  or  entirely  invisible  to  the  microscope. 

Ashburn  and  Craig, t  in  a  preliminary  report  on  their  re- 
searches into  the  etiology  of  dengue,  state  that  so  far  as  they  have 
gone  they  have  found  that  the  causative  agent,  whatever  its 
character,  resides  in  the  blood  of  persons  suffering  from  the 
disease,  since  intravenous  injections  of  human  beings  with 
blood  from  dengue  fever  patients  produce  the  disease.  The 
organism  is  probably  ultramicroscopic,  since  it  passes  through 
a  Pasteur  filter.  The  incubation  period  in  the  persons  in- 
jected is  four  days  and  it  is  the  same  whether  filtered  or  un- 
filtered  blood  is  employed.  The  disease,  they  state,  is  not  con- 
tagious, but  is  conveyed  by  at  least  one  species  of  mosquito, 
Culeoc  fatigans. 

They  find  on  further  study J  that  no  organism,  either  bacter- 
ium or  protozoon,  can  be  demonstrated  in  the  blood. 

Modes  of  Introduction. — There  are  various  avenues  by 
which  bacteria  may  enter  the  body  to  produce  disease.  In- 
fection of  the  embryo  through  the  ovum  or  semen  seems  to  be 
of  rare  occurrence.  Syphilis  is  transmitted  in  this  manner. 
The  embryo  may  be  infected  through  the  placenta,  although 
not  commonly.  'The  bacilli  of  typhoid  fever  and  the  pus- 
forming  bacteria  have  been  known  to  be  conveyed  through  it. 

*See  Reed  and  Carroll.  American  Medicine.  February  22,  1902.  For  an 
admirable  review  of  this  subject  see  Roux.  Sur  les  Microbes  dits  Invisibles. 
Bulletin  de  VInstitut  Pasteur.  Vol.  I.,  Nos.  i  and  2.  Also  Dorset.  Invisible 
Microorganisms.  United  States  Department  of  Agriculture,  Bureau  of  Animal 
Industry,  Circular  No.  57.  1904. 

•fjourn.  Am.  Medl  Assn.  V.,  XLVIII.     No.  8.,  FeU  23,  1907.  p.  693. 

\Journ.  Inject.  Dis.     No.  3,  Vol.  4.     June  15,  1907.     pp.  440-475. 


BACTERIA    IN    DISEASE.  173 

Tuberculosis  may  also  be  transmitted  through  the  placenta; 
how  frequently  is  still  uncertain.  Occasionally  the  exan- 
thematous  fevers  are  transmitted  from  the  mother  to  the  fetus. 

The  surfaces  covered  with  thick,  stratified  epithelium  are  not 
likely  to  be  penetrated  by  bacteria  except  through  some  wound 
or  other  lesion.  This,  for  instance,  is  true  of  the  skin,  the 
mouth,  the  vagina  and  bladder.  The  infection  of  bubonic 
plague  appears  to  be  introduced  most  often  by  means  of 
wounds  in  the  skin.  Bacteria  more  easily  penetrate  surfaces 
having  a  thin,  columnar  epithelium  such  as  occurs  in  the 
intestines,  the  middle  ear,  bronchi  and  bronchial  tubes,  uterus 
and  Fallopian  tubes. 

The  thin,  flat  epithelial  cells  of  the  air-vesicles  of  the  lungs, 
as  would  be  expected,  seem  to  be  passed  with  comparative  ease. 
On  epithelial  surfaces  covered  with  cilia,  as  in  the  bronchi  and 
bronchial  tubes,  the  Eustachian  tubes,  the  uterus  and  Fallopian 
tubes,  the  current  toward  the  exterior  created  by  the  cilia  acts 
beneficially  in  removing  bacteria. 

The  tonsils  and  lymph-follicles  of  the  intestines,  especially 
the  lymphoid  tissue  of  the  ileum  and  the  vermiform  appendix, 
are  points  where  bacterial  invasion  frequently  begins.  The 
lymphoid  tissue  of  the  appendix  may  have  some  influence  in 
predisposing  to  infection  at  that  point  and  to  appendicitis. 
On  the  other  hand,  it  is  certain  that  the  progress  of  many  in- 
fections is  checked  by  the  lymph-nodes.  That  is  repeatedly 
seen  in  the  ordinary  post-mortem  wound  where  the  spread  of 
the  inflammation  along  the  arm  is  checked  suddenly  at  the  el- 
bow or  axilla.  The  participation  of  the  lymphoid  structures 
in  most  infections  is  well  known.  How  far  this  is  a  conserva- 
tive process  it  is  impossible  to  say. 

In  most  cases  of  infectious  disease  a  point  of  entrance  for  the 
bacteria  may  be  discovered.  As  a  rule,  the  invading  microbes 
produce  a  lesion  at  the  point  where  they  are  introduced,  as  in 
the  familiar  cases  of  boils  and  carbuncles  when  pyogenic 


174  MANUAL    OF    BACTERIOLOGY. 

bacteria  enter  the  skin,  or  of  the  tubercles  found  in  the  lungs 
when  the  bacilli  lodge  in  the  respiratory  tract.  However, 
there  are  cases  of  septicemia  and  pyemia  in  which  the  most 
careful  search  fails  to  reveal  the  place  at  which  the  bacteria 
entered.  The  bacilli  of  plague  usually  produce  no  reaction 
at  the  point  of  entrance. 

Tubercle  bacilli  may  pass  through  thin  epithelial  surfaces 
and  lodge  in  the  deeper  structures  underneath,  where  they 
produce  definite  lesions  and  leave  no  trace  to  mark  the  point  of 
entrance.  For  example,  they  may  pass  by  the  lungs  and  enter 
the  bronchial  glands,  and  form  tubercles  in  that  situation. 

Ravenel  has  shown  that  they  pass  through  the  walls  of  the 
intestines  without  causing  any  lesion  there,  and  produce  tuber- 
culosis in  the  lungs  and  elsewhere. 

Experiments  on  animals  have  shown  that  bacteria  may  be 
very  rapidly  disseminated  after  their  introduction.  The  inocu- 
lation of  mice,  for  instance,  with  anthrax  bacilli  has  been  known 
to  prove  fatal,  although  the  wound  was  washed  immediately 
with  the  strongest  antiseptic  solutions  or  the  part  amputated 
within  a  few  minutes. 

The  agencies  concerned  in  the  transfer  of  infection  have  been 
referred  to  in  Part  II.,  Chapter  II?.  In  all  cases,  however, 
there  is  direct  or  indirect  connection  with  another  case  of  the 
same  disease.  W.  H.  Park  was  able  to  cultivate  diphtheria 
bacilli  from  bedclothing  soiled  by  the  expectoration  of  diph- 
theria cases.  Baldwin  has  shown  that  tubercle  bacilli  may  be 
found  on  the  hands  of  patients  having  pulmonary  tuberculosis, 
especially  those  who  expectorate  on  handkerchiefs.  Winslow 
found  bacillus  coli  communis  on  the  hands  of  5  per  cent,  to  19 
per  cent,  of  those  examined;  his  observations  suggest  the 
possibility  that  typhoid  bacilli  can  be  carried  in  a  similar 
manner. 

Direct  Contact. — Many  diseases  are  conveyed  from  a  sick 
individual  to  a  healthy  person  by  direct  contact  as  in  gonor- 


BACTERIA   IN    DISEASE.  1 75 

rhea  and  syphilis.  This  mode  of  transfer  is  probably  quite 
frequent  in  many  diseases. 

Healthy  Persons  as  Carriers  of  Infection. — There  are 
numbers  of  examples  of  infection  having  been  spread  by 
healthy  persons  who,  though  not  suffering  themselves,  never- 
theless harbor  the  infectious  agent.  This  matter  was  promi- 
nently brought  to  light  in  the  cholera  epidemic  in  Hamburg, 
Germany,  in  1892,  where  a  number  of  persons  who  passed 
quarantine,  and  traveled  to  other  parts  of  Germany  were  found 
to  have  the  cholera  spirillum  in  their  feces.  In  some  cases 
local  outbreaks  were  traced  to  these  persons.  Other 
infectious  diseases  have  been  traced  to  the  same  source. 
Soper*  has  published  the  history  of  a  case  in  which  a  healthy 
individual  carried  and  disseminated  the  typhoid  bacillus  for 
years.  Other  cases  of  the  same  kind  have  been  reported. 
Dehlerf  reports  the  case  of  a  man  who  was  a  constant  typhoid 
bacillus  carrier  on  whom  he  operated  for  the  sole  purpose  of 
cleaning  out  the  gall-bladder  to  rid  it  of  the  typhoid  bacilli. 
He  afterward  operated  an  another  similar  case  for  the  same 
purpose,  with  the  result  that  the  bacilli  disappeared  from  the 
stools.  In  regard  to  diphtheria  there  is  abundant  evidence 
that  healthy  persons  may  harbor  the  organism. 

Air. — Excepting  under  certain  special  conditions,  the  air 
does  not  contain  the  germs  of  disease.  Dried  pulverized 
sputum  containing  tubercle  bacilli  may  float  in  the  atmosphere 
as  dust.  Flligge  found  that  powerful  expiratory  efforts  like 
coughing  and  sneezing  may  carry  tubercle  bacilli  with  small 
particles  of  secretion  into  the  air,  and  that  they  may  remain  in 
suspension  some  time.  The  pus-producing  bacteria  may  be 
present  in  dust.  Infectious  elements  which  happen  to  be 
present  in  the  air  are  usually  attached  to  particles  of  dust. 


*  Jouru.  Am.Med.  Assn.     June  13,  1907.     p.  2019. 

t Munch  Med.  Wochenschr     Oct.  22,  1907.     Abst.  Journ.  Am.  Med  Assn. 
Nov.  30,  1907.     Also  Eben.     July  6,  1907,  p.  94. 


176  MANUAL    OF    BACTERIOLOGY. 

Wool-sorter's  disease  is  a  name  sometimes  applied  to  anthrax 
in  man  when  acquired  by  inhaling  the  dust  from  wool  which 
contains  the  anthrax  bacilli  or  spores. 

The  atmosphere  in  the  vicinity  of  cases  of  the  exanthema- 
tous  fevers  must  necessarily  at  times  contain  the  germs  of  these 
diseases. 

Water  is  commonly  regarded  as  the  usual  medium  for 
the  transmission  of  the  infection  in  typhoid  fever,  and  Asiatic 
cholera,  and  probably  all  forms  of  dysentery  besides  various 
nondescript  disturbances  of  the  alimentary  tract. 

Milk  from  tuberculous  cows  may  carry  the  bacilli  of  tubercu- 
losis as  already  stated;  this  is,  of  course,  of  the  utmost  im- 
portance in  the  case  of  young  infants.  Typhoid  fever  and 
cholera,  and  probably  dysentery,  scarlet  fever  and  diphtheria 
would  all  appear  to  be  diseases  which  might  be  conveyed 
through  the  medium  of  milk  and  in  some  cases  of  these  diseases 
this  mode  of  infection  has  been  quite  clearly  demonstrated. 
Not  only  milk  but  all  other  forms  of  uncooked  food  may  serve 
as  carriers  of  infection  to  the  intestines. 

The  Soil  is  of  importance  as  a  mode  of  conveyance  of  in- 
fection because  of  the  frequent  presence  in  it  of  the  bacteria  of 
tetanus  and  of  malignant  edema.  Bacillus  aerogenes  capsulatus 
may  occur  in  the  soil,  and  may  infect  dirty  wounds.  The 
spores  of  anthrax  bacilli  are  present  in  the  soil  of  certain  locali- 
ties, and  may  produce  anthrax  in  cattle. 

Flies.* — Under  suitable  conditions,  flies  play  an  important 
part  in  transporting  the  bacteria  of  cholera  and  typhoid  fever 
from  the  excreta  of  these  diseases  to  food  substances,  which  they 
may  contaminate.  Flies  which  have  access  to  tuberculous 
sputum  may  deposit  tubercle  bacilli  on  food.f  Buchanan  J 
has  shown  that  the  common  house-fly  and  the  blue-bottle  fly 

*Nuttall.  Role  of  Insects,  etc.,  in  Disease.  John  Hopkins  Hospital  Reports. 
Vol.  VIII.  1900. 

fLord.     Boston  Medical  and  Surgical  Journal.     Dec.  15,  1904. 
^Circular  71,  U.  S.  Department  of  Agriculture,  Bureau  of  Entomology. 


BACTERIA    IN    DISEASE.  177 

are  capable  of  carrying  bacteria  on  their  feet.  The  organisms 
experimented  with  were  typhoid,  swine  fever,  staphylococcal 
afiscess,  pulmonary  tuberculosis  and  anthrax.  L.  O.  Howard* 
and  many  others  before  and  since  have  pointed  out  the  same 
source  of  danger  of  spreading  of  infection.  To  what  extent 
diseases  are  disseminated  by  fleas,  bedbugs  and  similar  crea- 
tures is  still  uncertain. 

In  this  connection  it  is  proper  to  refer  to  certain  diseases  due 
to  animal  microorganisms.  Malaria  is  conveyed  from  man  to 
man  by  mosquitoes  of  the  genus  Anopheles,f  and  is  probably 
transmitted  exclusively  in  this  manner.  The  parasite  of 
malaria  undergoes  part  of  its  cycle  of  development  in  man, 
and  another  part  in  the  mosquito.  Similarly,  in  Texas  fever,  a 
disease  of  cattle,  it  has  been  shown  by  Theobald  Smith  that  the 
parasite  (a  protozoon,  Pirolasma)  passes  from  cow  to  cow 
through  the  cattle-tick  (Bophilus  annulatus  or  bovis).J  In 
surra,  a  disease  chiefly  affecting  horses,  and  in  the  tsetse-fly 
disease  of  animals  the  parasite  (a  protozoon,  Trypanosoma)  is 
transmitted  by  the  bites  of  flies. §  It  has  been  shown  that  the 
infectious  agent  of  yellow  fever  may  be  introduced  into  man  by 
mosquitoes  of  the  genus  Stegomyia.||  Under  the  administra- 
tion of  the  United  States  army  yellow  fever  was  suppressed  in 
Havana  chiefly  by  measures  intended  to  prevent  the  disease 
from  being  carried  by  mosquitoes.  Equally  good  results  have 
since  been  attained  in  controlling  an  epidemic  of  yellow  fever 
at  Laredo,  Texas,  in  1903,^  and  a  great  reduction  in  the 
mortality  at  Rio  Janeiro,  Brazil,  has  been  effected. 

Auto-infection. — It  is  possible  for  the  bacteria  of  a  disease 
which  is  localized  in  one  part  of  the  body  to  be  conveyed  to 

*Lancet.     July  27,  1907.     Abstract  in  Journ.  Am.  Med.  Assn.,  Aug.  24,  1907. 
fFor  detail  concerning  mosquitoes  consult  the  book  of  Dr.  L.  O.  Howard. 
McClure,  Phillips   &  Co. 

|See  V.  A.  Moore.     Infectious  Diseases  of  Animals.     1902. 
§  Report  on  Surra.     U.  S.  Bureau  of  Animal  Industry.     1902. 
| (Carroll.     Journal  American  Medical  Association.     May  23,  1903. 
^[Guiteras.     Journal  American  Medical  Association.     July,  9,   1904. 
12 


178  MANUAL    OF    BACTERIOLOGY. 

another  part  of  the  body,  and  to  cause  the  disease  in  the  new 
situation  in  the  individual's  own  body.  This  is  called  auto- 
infection.  It  is  also  the  case  that  certain  bacteria  remain 
inert  when  present  in  certain  parts  of  the  body,  but  cause  dis- 
ease when  transferred  to  other  parts.  The  microbes  of  lobar 
pneumonia,  for  instance,  flourish  in  the  mouths  of  a  large 
number  of  people  and  under  favoring  circumstances  may 
produce  disease  in  the  lungs  or  other  parts,  though  it  is  not 
known  in  this  case  whether  the  infecting  organism  comes  from 
the  patient's  own  mouth  or  from  the  outside.  The  bacillus 
coli  communis,  which  constantly  inhabits  the  intestines,  may 
invade  other  organs  and  exhibit  pathogenic  properties  when 
the  way  is  opened  up  for  it  by  other  disease  processes.  Persons 
suffering  from  gonorrhea  frequently  infect  their  eyes  by  trans- 
fering  the  urethral  secretion. 

Bodily  Conditions  that  Predispose  to  Infection.— The 
development  of  an  infectious  disease  may  be  favored  by  certain 
bodily  conditions  spoken  of  as  predisposing  causes.  These 
may  be  general  and  operate  in  such  a  way  as  to  lowrer  the 
general  tone  of  the  body,  as  it  is  rather  vaguely  stated;  or  may, 
in  addition,  predispose  to  certain  definite  infection.  Thus 
hunger,  cold  and  exhaustion  make  the  body  more  liable  to  the 
inroads  of  pathogenic  bacteria  in  general;  so  also  do  anemia 
and  chronic  diseases.  As  an  example  of  predisposition  to 
specific  infection  is  the  well-known  fact  that  those  suffering 
from  diabetes  are  espiecially  liable  to  infection  by  the  pus- 
producing  bacteria  and  the  bacillus  tuberculosis.  Prolonged 
anesthesia  probably  renders  patients  who  have  undergone 
operations  more  liable  to  surgical  infections  and  to  absorption 
of  bacterial  poisons.  Predisposition  to  infection  may  also 
arise  in  such  cases  from  auto-intoxication  with  the  products  of 
(disordered  metabolism  of  the  patient's  own  cells.  Some  of  the 
above-mentioned  conditions  can  be  imitated  in  laboratory  ex- 
periments. Hens  in  a  normal  condition  are  not  susceptible  to 


BACTERIA    IN    DISEASE.  179 

the  anthrax  bacillus,  but  Pasteur  succeeded  in  making  them 
susceptible  to  this  disease  by  artificially  cooling  them.  Frogs, 
on  the  other  hand,  which  also  are  resistant  to  anthrax,  may  be 
made ,  susceptible  by  keeping  them  at  an  abnormally  high 
temperature.  Rats  were  made  more  susceptible  to  anthrax 
by  physical  exhaustion  produced  by  making  them  run  a  tread- 
mill and  pigeons  by  starvation. 

Abbott  found  "that  the  normal  vital  resistance  of  rabbits  to 
infection  by  streptococcus  pyogenes  is  markedly  diminished 
through  the  influence  of  alcohol,  when  given  daily  to  the  stage 
of  acute  intoxication."  This  effect  of  alcohol  was  evident  to  a 
marked  degree  with  the  anthrax  bacillus,  but  it  was  less  notice- 
able for  bacillus  coli  communis,  and  not  observed  for  staphylo- 
coccus  pyogenes  aureus. 

Climate  and  altitude  appear  to  influence  the  liability  to 
infection  with  the  tubercle  bacillus;  for,  as  everyone  knows, 
tuberculous  affections  occur  less  commonly  in  elevated  regions 
than  in  lower  and  more  densely  populated  districts. 

There  are  probably  a  great  many  other  as  yet  obscure  con- 
ditions affecting  predisposition  to  infection. 

Age. — In  general,  infants  are  more  susceptible  to  infections 
than  adults,  though  apparently  nearly  exempt  from  the  exan- 
thematous  fevers  during  the  early  weeks  of  life.  Osteomye- 
litis is  commoner  in  infants  than  in  adults,  as  also  is  tubercu- 
lous meningitis. 

Individual  Predisposition. — The  influence  of  individual 
predisposition  is  often  very  marked;  though,  as  Welch  says, 
"The  fact  that  some  individuals  are  attacked  and  others,  ap- 
parently equally  exposed  to  the  danger  of  infection,  escape, 
is  not  always  due  to  any  especial  predisposition  on  the  part  of 
the  former.  It  may  be  that  the  germs  hit  the  one  and  miss  the 
other,  and  we  would  have  no  more  right  to  say  that  the  former 
are  especially  predisposed  than  to  say  that  those  who  fall  in 
battle  are  predisposed  to  bullets  and  those  who  escape  are 


l8o  MANUAL    OF    BACTERIOLOGY. 

bullet-proof."  Nevertheless,  as  was  clearly  shown  in  the 
cholera  epidemic  in  Hamburg,  Germany,  in  1892,  many  per- 
s)ns  were  found  who  harbored  the  comma  bacillus  in  their  in- 
testines without  exhibiting  any  symptoms  of  the  disease. 
Many  healthy  persons  have  been  found  to  harbor  the  diph- 
theria bacillus  in  their  throats.  Pneumococci  are  present  in  the 
mouths  of  many  healthy  individuals.  From  these  and  other 
examples  which  might  be  cited,  it  is  apparent  that  individual 
resistance  and  individual  predisfusition,  due  to  causes  as  yet 
obscure,  are  important  factors  in  infectious  diseases. 

It  is  probable  that  the  importance  of  an  hereditary 
tendency  to  certain  infections,  notably  tuberculosis,  has  been 
overrated. 

Predisposition  of  Different  Organs  and  Tissues.— Cer- 
tain tissues  of  the  body  are  liable  to  attack  from  one  kind  of  or- 
ganism, while  another  tissue  maybe  more  susceptible  to  invasion 
by  a  different  species  of  bacterium.  The  mucous  membrane 
of  the  urethra  is  specially  open  to  attack  from  the  gonococcus; 
that  of  the  intestines  from  the  dysentery  bacillus,  the  cholera 
spirillum  and  the  typhoid  bacillus;  the  lungs  are  more  liable  to 
attack  from  the  tubercle  bacillus  than  other  organs;  the  eye 
may  be  attacked  by  a  large  number  of  different  organisms* 
beside  being  subject  to  attack  of  a  special,  possibly  of  two  or 
more  special  bacteria.  Conjunctivitis  has  been  found  to 
be  caused  by  the  pneumococcus,  the  diphtheria  bacillus, 
streptococcus,  staphylococcus,  gonococcus,  B.  coli  communis, 
meningococcus  intercellularis,  several  of  the  group  of  organ- 
isms to  which  the  Friedlander's  pneumococcus  belongs,  B. 
influenzae.  In  addition  to  these  organisms,  a  special  bacterium, 
the  Koch- Weeks  bacillus,  has  been  found  to  cause  epidemic 
conjunctivitis  in  all  lands.  This  is  a  fine  bacillus  much  resem- 
bling that  of  mouse  septicemia. 

Race. — The  influence  of  racial  predisposition  is  undeniable. 

*Axenfeld  in  Kolle  and  Wassermann.     Bd.  III.,  1903.     pp.  489-575. 


BACTERIA    IN    DISEASE.  l8l 

For  example,  it  is  known  that  the  negro  race  is  much  less  sus- 
ceptible to  yellow  fever  than  the  white  race. 

Local  Conditions. — A  most  important  influence  in  de- 
termining the  occurrence  of  infections  may  be  found  in  local 
bodily  conditions.  In  endocarditis  the  lesion  usually  occurs 
along  the  line  of  closure  of  the  heart-valves,  indicating  that  the 
point  subjected  to  the  greatest  friction  is  the  part  of  the  en- 
docardium most  liable  to  infection.  Regions  where  there  is 
passive  hyperemia  are  more  vulnerable,  as  is  seen  in  hypostatic 
pneumonia;  but  on  the  other  hand  v.  Bier  has  found  that 
the  production  of  passive  hyperemia  by  artificial  means  tends 
to  bring  about  a  rapid  improvement  in  local  infections. 
Localities  which  have  suffered  from  previous  inflammation  or 
irritation  are  rendered  more  liable  to  subsequent  infection,  as 
when  the  bladder  or  pelvis  of  the  kidney  containing  a  calculus 
becomes  the  seat  of  a  suppurative  cystitis  or  pyelitis. 

Local  conditions  become  of  great  importance  in  surgery. 
The  surgeon  can  seldom  be  certain  of  dealing  with  a  perfectly 
aseptic  wound,  and  must  rely  to  a  large  extent  upon  the  power 
inherent  in  the  fluids  and  tissues  to  prevent  the  development  of 
bacteria.  It  is  important,  therefore,  to  keep  the  resisting 
power  of  the  tissues  at  the  highest  possible  point.  Injury  of 
the  tissues  disposes  the  part  to  infection;  so  do  strangulation  and 
necrosis.  In  operating,  it  is  to  be  remembered  that  hyperemic 
and  edematous  parts  are  more  likely  to  become  infected;  so 
also  are  anemic  regions.  An  infarct  of  the  lung  which  was 
originally  sterile  may  be  infected  with  bacteria  through  in- 
halation, and  undergo  suppuration  or  gangrene.  The  pres- 
ence of  foreign  bodies  in  the  tissues  disposes  to  infection.  In- 
jection of  the  staphylococcus  pyogenes  aureus  into  a  rabbit's 
tissues  is  not  always  followed  by  suppuration,  but  if  a  foreign 
body,  like  a  piece  of  sterilized  potato,  be  inserted  at  the  same 
time,  infection  is  much  more  likely  to  occur.  When  lesions  are 
produced  in  the  internal  viscera  of  animals  by  cauterization  or 


1 82  MANUAL    OF    BACTERIOLOGY. 

crushing  and  bacteria  then  injected  subcutaneously  or  into  the 
blood,  the  bacteria  lodge  in  the  lesions  and  multiply.* 

Amount  of  Infectious  Material.— A  large  number  of  bac- 
teria introduced  into  the  body  simultaneously  will  be  more 
likely  to  produce  infection  than  a  small  number.  This  factor 
is  of  less  importance  with  organisms  whose  virulence  is  very 
constant  than  with  those  of  more  variable  virulence. 

Variability  in  the  Virulence  of  Bacteria. — The  occur 
rence  of  an  infectious  disease  depends  very  largely  upon  the 
virulence  of  the  bacteria.  Any  species  of  pathogenic  bacteria 
may  vary  in  virulence  at  different  times.  In  some  cases  the 
virulence  is  not  easily  lost,  as  with  the  anthrax  bacillus;  in 
others  the  virulence  is  maintained  in  cultures  only  with  dif- 
ficulty, as  in  the  case  of  the  micrococcus  lanceolatus  (of 
pneumonia]  and  the  streptococcus  pyogenes.  As  a  rule,  the 
virulence  is  likely  to  be  diminished  in  old  cultures.  It  may 
sometimes  be  preserved  better  in  the  ice-chest  than  at  the  room 
temperature.  The  virulence  of  the  anthrax  bacillus  becomes 
diminished  if  it  is  cultivated  at  42°  to  43°  C.  Exposure  to  light 
and  to  oxygen  tends  to  weaken  the  virulence;  and  also  cultiva- 
tion upon  unfavorable  media,  such  as  those  containing  a 
small  proportion  of  carbolic  acid  01  certain  other  chemical 
germicides. 

In  laboratory  work  the  virulence  is  usually  maintained  best 
by  inoculating  the  bacteria  from  time  to  time  into  susceptible 
animals.  Bacteria  coming  freshly  from  infected  animals  are 
likely  to  be  highly  virulent.  The  virulence  may  be  increased 
by  beginning  with  an  especially  sensitive  animal  like  a  very 
young  guinea-pig,  and  progressively  inoculating  into  less  sensi- 
tive animals.  The  infection  of  relatively  insusceptible  animals 
may  sometimes  be  produced  by  the  injection  of  a  very  large 
dose  of  the  bacteria.  The  addition  of  the  toxic  products  of  the 
bacteria,  which  may  be  obtained  by  using  large  doses  of  cul- 

*Cheesman  and  Meltzer.     Journal  Experimental  Medicine.     Vol.  III. 


BACTERIA    IN    DISEASE.  183 

lures  in  bouillon,  makes  infection  more  likely.  Cultivation  on 
a  particular  medium  may  maintain  or  increase  the  virulence. 

Finally,  the  combination  of  two  or  more  kinds  of  bacteria 
may  produce  infection  when  neither  one  would  do  so  alone. 
On  the  other  hand,  it  is  said  that  the  fatal  effects  of  an  inocula- 
tion of  virulent  anthrax  bacilli  into  a  susceptible  animal  may 
be  averted  if  the  animal  be  inoculated  with  a  culture  of  bacillus 
pyocyaneus  shortly  afterward. 

Mixed  Infection. — It  is  not  uncommon  in  disease  to  find 
two  kinds  of  bacteria  associated  together,  producing  a  mixed 
infection.  In  diphtheria,  very  frequently,  the  bacillus  of 
diphtheria  is  found  to  be  accompanied  in  the  membrane  by  the 
streptococcus  pyogenes.  The  course  of  the  diphtheria  may  be 
modified  in  this  manner.  The  term  secondary  injection  is 
rather  loosely  used.  It  is  sometimes  employed  to  designate  an 
infection  occurring  in  an  individual,  the  resisting  power  of  whose 
tissues  has  been  weakened  by  some  chronic  organic  disease. 
Such  an  infection  is  often  called  a  terminal  injection.  Terminal 
infections  are  very  common  in  cases  of  carcinoma,  chronic 
nephritis,  arteriosclerosis,  and  in  many  other  diseases. 

Concerning  terminal  infections  Osier  says:  "It  may  seem 
paradoxical,  but  there  is  truth  in  the  statement  that  persons 
rarely  die  of  the  disease  with  which  they  suffer.  Secondary 
infections,  or,  as  we  are  apt  to  call  them  in  hospital  work, 
terminal  infections,  carry  off  many  of  the  incurable  cases  in 
the  wards." 

The  term  secondary  injection  is  also  used  for  the  modification 
of  an  infectious  process  which  has  been  in  existence  for  some 
time,  by  infection  with  a  second  species  of  bacteria.  That 
takes  place,  for  instance,  in  pulmonary  tuberculosis,  when 
the  invasion  of  the  already  tuberculous  lungs  by  the  pyogenic 
micrococci  assist  in  the  formation  of  cavities.  In  this  sense 
it  will  be  seen  that  the  term  secondary  infection  is  used  as  a 
name  for  a  variety  of  mixed  infection.  In  the  secondary, 


184  MANUAL    OF    BACTERIOLOGY. 

mixed  and  terminal  infections,  the  bacteria  which  enter  second- 
arily are  likely  to  be  of  the  pus-producing  varieties,  especially 
the  streptococcus  pyogenes. 

As  to  the  mechanism  which  bacteria  make  use  of  in  order 
to  produce  disease,  according  to  our  present  knowledge,  they 
work  chiefly  through  the  poisonous  substances  formed  by 
them  and  deposited  in  the  bodies  of  the  persons  suffering  from 
the  disease.  The  theory  that  bacteria  have  an  important  in- 
fluence through  the  destruction  of  substances  taken  by  them 
from  the  bcdy  of  the  patient  for  food  is  no  longer  entitled  to 
much  weight;  neither  are  we  able  in  most  cases  to  account 
for  the  pheriomena  of  disease  by  any  mechanical  action  on  the 
part  of  the  bodies  of  bacteria.  That  such  action  does  occa- 
sionally take  place  may  be  seen  in  experimental  anthrax  in 
mice,  where  the  blood-capillaries  of  the  liver  and  kidneys  may 
be  completely  plugged  with  masses  of  anthrax  bacilli.  The 
diseases  in  which  the  circulating  blood  is  swarming  with 
bacteria  are  much  commoner  in  the  lower  animals  than  in  man. 

Toxemia. — By  toxemia  is  ment  the  absorption  of  poisonous 
bacterial  products  from  a  localized  point  of  invasion,  and  their 
dissemination  throughout  the  body  by  means  of  the  circulation. 
We  see  typical  toxemias  in  diphtheria  and  tetanus.  In  surgery 
the  term  sapremia  is  used  to  cover  a  similar  condition  of  affairs 
when  the  absorption  proceeds  from  a  wound  or  denuded  sur- 
face, •  as  may  happen  in  the  puerperal  uterus. 

Septicemia. — In  septicemia  there  is  not  only  absorption 
of  bacterial  poisons,  but  an  invasion  by  bacteria  of  the  living 
tissues  and  the  blood.  The  presence  of  large  numbers  of 
bacteria  disseminated  throughout  the  body  and  in  the  blood  is 
less  common  in  septicemia  in  man  than  in  such  diseases  as 
anthrax  in  the  lower  animals.  Typical  septicemias  in  man 
are  found  in  relapsing  fever  and  certain  cases  of  bubonic 
plague.  For  pyemia,  see  the  article  on  Suppuration,  Part  IV. 

The  principal  agencies  in  effecting  recovery  from  infectious 


BACTERIA   IN   DISEASE.  185 

diseases  are  the  destruction  of  the  bacteria  by  the  cells  of  the 
body  (phagocytosis),  the  development  of  new  substances 
which  neutralize  their  action  (antitoxins)  and  the  presence  or 
formation  in  the  body  of  substances  which  destroy  bacteria 
(lysins).  These  phenomena  are  discussed  in  the  chapter  on 
Immunity.  A  factor  of  less  importance  is  the  elimination  of 
bacteria  by  the  excretory  organs.  Investigators  who  have 
made  experiments  on  animals  disagree  as  to  whether  or  not  the 
bacteria  which  have  been  injected  into  the  body  appear  in  the 
urine  before  they  have  damaged  the  structure  of  the  kidney. 
The  extent  to  which  the  excretory  organs  act  in  eliminating 
bacterial  toxins  is  not  yet  known.  Some  bacteria,  as  has 
already  been  stated,  may,  in  the  end,  produce  substances  that 
are  inimical  to  their  own  growth. 


OF   THE 

(  UNIVERSITY  ) 

OF 


CHAPTER  VI. 
BACTERIAL  POISONS.* 

There  are  now  recognized  three  different  kinds  of  bacteria 
poisons:  (a)  Ptomaines,  or  poisons  produced  by  bacteria  ou 
of  the  medium  upon  which  they  grow;  (b)  endotoxions,  o 
poisons  contained  within  the  bacterial  cell  and  liberated  only 
upon  the  disintegration  of  the  cells;  (c)  toxins,  or  poisons  liber 
ated  into  the  culture-medium  and  into  the  animal   body  by 
the  bacteria  during  their  growth,  probably  as  a  sort  of  excretory 
product.     The  ptomaines  are  not  specific,  •  they  are  not  pro 
duced  necessarily  by  pathogenic  bacteria,  but,  as  the  nam 
implies,  may  be  developed  in  putrefying  meat.     These  bodie 
were  first  studied  by  Brieger.     They  are  crystalline  in  charac 
ter.     On  the  other  hand,  it  is  now  generally  accepted  that  ii 
most,  if  not  all  of  the  infectious  diseases,  the  principal  symptom 
and  lesions  are  to  be  attributed  to  the  action  of  either  endo- 
toxins  or  of  toxins.     Even  in  those  cases  where  poisonous  sub- 
stances are  not  demonstrable  in  cultures  or  demonstrable  with 
difficulty  only,  there  is  reason  to  believe  that  the  bacteria  in 
such  cases  may,  nevertheless,  produce  poisons  in  the  animal 
body. 

Certain  infectious  diseases  afford  examples  of  poisoning  by 
bacterial  products  in  an  extremely  marked  manner.  In  teta- 
nus the  local  wound  may  be  trifling  and  in  itself  utterly  inca- 
pable of  giving  rise  to  the  violent  muscular  spasms  from  which 
the  patient  suffers  in  consequence  of  the  powerful  poison  which 
the  tetanus  bacillus  forms  at  the  point  of  infection.  In  diph- 

*For  a  full  consideration  of  this  subject  see  Vaughan  and  Novy.  The  Cellu- 
lar Toxins.  1902. 

1 86 


BACTERIAL   POISONS.  187 

theria,  although  the  condition  in  the  throat  may  be  one  of  severe 
inflammation,  it  is  of  itself  insufficient  to  explain  the  profound 
prostration  and  other  symptoms  of  general  poisoning  which 
the  case  manifests. 

Bacterial  poisons  may  be  diffused  through  the  culture- 
medium  or  they  may  be  retained  in  the  bodies  of  the  bacteria. 
Consequently,  they  are  classed  as  extracellular  and  intracell- 
ular.  Intracellular  poisons  are  called  endotoxins.  In  cultures 
of  the  diphtheria  and  of  the  tetanus  bacilli  the  culture-medium 
contains  the  poison,  and  injections  of  the  broth  in  which  these 
organisms  have  been  grown  produce  these  diseases  just  as 
promptly  and  effectually  as  injections  of  the  bacteria  themselves. 
Even  when  the  bacteria  in  these  cultures  are  entirely  removed 
by  nitration  through  porcelain  filters,  the  nitrate  reproduces  the 
diseases  with  all  their  symptoms  just  as  characteristically  as 
the  unfiltered  cultures.  The  toxin  from  the  diphtheria  bacillus 
and  that  from  the  tetanus  bacillus  are  therefore  extracellular 
toxins,  and  these  two  are  the  only  ones  which  are  extracellular 
as  far  as  is  yet  known.  On  the  other  hand,  endotoxins  are  not 
liberated  into  the  culture-medium.  They  are  only  set  free  by 
breaking  up  the  cells,  either  mechanically,  by  grinding  in  a  mor- 
tar, or  by  disintegration  in  some  other  way.  The  disintegration 
of  these  bacteria  in  the  animal  body  is  probably  the  way  in 
which  certain  of  them  cause  disease.  Typhoid  bacilli  and  chol- 
era spirilla  probably  act  in  this  way. 

Tracy*  obtained  a  strong  toxin  by  disintegrating  B.  prodigio- 
sus  in  various  ways. 

It  seems  probable  that  some  bacteria  do  not  produce  either 
intra-  or  extracellular  toxins  in  the  strict  meaning  of  the  word. 
Anthrax  bacilli  and  tubercle  bacilli  are  of  this  sort. 

The  first  bacterial  poisons  to  be  studied  thoroughly  were 
the  ptomaines.  The  observation  that  the  poisonous  effects 
which  follow  the  injection  into  animals  of  certain  ptomaines 

*Journ.  Med.  Research.     Vol.  XVI.,  No.  2,  1907.     pp.  307-327. 


MANUAL    OF   BACTERIOLOGY. 


derived  from  bacterial  cultures,  suggested  the  idea  that  simila 
ptomaines,  formed  by  the  action  of  bacteria  in  the  livinj 
body,  might  account  for  the  symptoms  of  many  of  the  infec 
tious  diseases.  The  ptomaines  were  most  readily  studiec 
because  of  the  comparative  facility  with  which  they  could  b 
isolated  in  a  condition  of  purity,  where  their  exact  chemica 
nature  could  be  determined.  They  were  found  to  be  basic 
compounds  derived  from  nitrogenous  material. 

A  similar  group  of  substances  called  leucomaines  has  been 
discovered,  which  are  formed  within  the  body  by  faulty 
metabolism  and  not  by  the  action  of  the  bacteria. 

Further  study  demonstrated,  however,  that  the  characteristic 
features  of  the  infectious  diseases  are  not  due  to  ptomaines,  but 
to  toxins.     The  term  toxin,  unless  otherwise  specially  desig- 
nated, applies  to  extracellular  toxins,  of  which  the  toxin  of 
diphtheria  and  that  of  tetanus  are  the  types;  indeed,  the  only 
representatives  so  far  obtained  are  from  bacteria,  though  there 
are  other  bodies  of  a  similar  nature  found  in  certain  plants 
and  in  snake  and  other  venom  from  animals.    Toxins  have 
not    been    obtained    yet  in  a  pure  state,   and  consequently 
their   exact   chemical   character   has   not   been    determined- 
but  much  has  been  learned  in  regard  to  their  physiological 
action,  and  more  information  in  this  direction  is  constantly 
being   obtained    by   experiments.     They  have    very  marked 
characteristics  and  they  do  not  act  like  ordinary  poisons,  but 
behave  as  ,f  they  had  the  power  of  reproduction.     An  ordinary 
poison,  such  as  arsenic,  strychnia  and  the  like,  begins  to  act  as 
soon  as  it  is  absorbed-there  is  no  period  of  incubation.     The 
toxins,  on  the  contrary,  have  a  distinct  period  of  incubation 
f  an  animal  is  given  a  fatal  dose  of  arsenic  or  strychnia,  it 
succumbs  within  a  comparatively  short  time;  it  is  at  most  a 
matter  of  a  few  hours.     But  if  an  animal  is  injected  with  a  fatal 
dose  of  the  toxin  of  tetanus  it  takes  some  time,  often  several 
days,  before  anv  symptoms  develop,  and  moreover  the  ani- 


BACTERIAL    POISONS.  189 

mal  may  remain  alive  for  days  afterward.  In  some  respects 
the  toxins  resemble  the  physiological  ferments,  ptyaline,  pepsin, 
and  the  like;  but  they  differ  from  these  in  that  the  physio- 
logical ferments  are  not  themselves  used  up  in  the  process  of 
fermentation,  whereas  the  toxins  are  used  up  in  the  production 
of  disease.  After  starch  has  been  converted  into  sugar  by 
ptyaline,  the  ptyaline  may  be  recovered  and  used  over  and  over 
again  to  convert  more  starch;  but  after  tetanus,  toxin  has  pro- 
duced tetanus  in  an  animal  it  cannot  be  recovered,  since  it  has 
become  firmly  united  to  the  cells.  Toxins,  therefore,  are  very 
peculiar  bodies,  behaving  like  ferments  in  requiring  consider- 
able time  to  produce  effects,  and  acting  like  unorganized  poi- 
sons in  being  used  up  in  the  tissue  changes  which  they  produce. 
Certain  substances  derived  from  the  vegetable  kingdom  behave 
in  the  same  manner  as  bacterial  toxins;  ricin,  abrin  and  robin 
are  examples  of  these.  The  poisons  of  scorpions  and  snakes 
are  also  poisons  which  act  like  toxins.  Other  properties  of 
toxins  will  be  considered  in  connection  with  antitoxin. 

Although,  as  has  been  stated/  the  toxins  have  not  been 
isolated  in  a  pure  condition,  they  have,  nevertheless,  been  ob- 
tained in  some  cases  in  an  extremely  concentrated  form. 
Brieger  and  Cohn  obtained  a  toxin  from  tetanus  bacilli  of 
which  o.ooopoop5  gram  killed  a  mouse  weighing  15  grams. 
Roux  and  Yersin  obtained  a  toxin  from  diphtheria  bacilli  of 
which  0.00005  gram  was  capable  of  killing  a  guinea-pig.  These 
figures  indicate  the  extremely  poisonous  character  of  these 
toxins.  Such  properties  permit  bacteria  growing  in  a  compara- 
tively limited  area  to  act  upon  parts  of  the  body  remote  from 
the  focus  of  infection. 

A  curious  and  unexplained  effect  of  some  toxins  is  the  production  of  minute 
areas  of  necrosis  in  certain  viscera,  as  the  liver.  Such  "focal  necroses"  have 
been  observed  to  be  formed  by  the  poisons  of  the  baiclli  of  diphtheria,  of  typhoid 
fever,  and  of  the  Micrococcus  lanceolatus  of  pneumonia,  and  following  the  injec- 
tion of  abrin  and  ricin. 

Besides  the  extracellular  toxins  produced  by  the  bacilli  of  diphtheria  and 


MANUAL    OF    BACTERIOLOGY. 

of  tetanus,  endotoxins,  have  been  obtained  from  the  spirillum  of  cholera,  the 
bacillus  of  typhoid  fever,  the  Bacillus  coli  communis,  the  bacillus  of  bubonic 
plague,  Bacillus  pyocyaneus;  Streptococcus  pyogenes,  and  Staphylococcus  py- 
ogenes  aureus.  The  extract  from  cultures  of  tubercle  bacilli,  called  tuberculin, 
and  that  from  glanders  bacilli,  called  mallein,  will  be  spoken  of  in  connection 
with  the  bacteria  themselves.  Besides  these  toxic  substances,  lysins  or  bodies 
which  disintegrate  bacteria  and  other  substances  have  been  obtained  from  bac- 
terial cultures.  Thus  pyocyanase  disintegrates  many  kinds  of  bacteria  (see  p. — ). 
Some  bacteria  seem  to  contain  lysins  which  disintegrate  the  bacteria  themselves 
in  which  they  are  produced — so-called  autolysins.  Vaughan  *  has  succeeded 
in  cultivating  anthrax  bacilli,  colon  bacilli,  and  other  bacteria  on  large  surfaces 
of  solid  media,  so  as  to  secure  quantities  of  the  bacterial  cells  sufficient  for  exten- 
sive chemical  tests.  The  toxin  of  the  colon  bacillus  proved  to  be  a  very  stable 
substance,  and  resistant  to  heat.  Most  toxins  become  inactive  at  comparatively 
low  temperatures  (60°  to  70°  C.). 

The  endotoxin  of  plague  f  is  destroyed  by  70°  C. ;  that  of  dysentery  by  80°  C., 
that  of  typhoid  by  127°  C. 

Other  physiological  properties  of  the  toxins  will  be  brought  out  in  connection 
with  the  discussion  of  immunity. 

There  is  good  reason  on  both  clinical  and  experimental  grounds  to  believe 
that  toxic  substances  are  formed  by  the  Micrococcus  lanceolatus  of  pneumonia. 

In  connection  with  bacterial  poisons  another  class  of  bodies 
may  be  conveniently  described;  these  are  agglutinins,  lysins 
and  precipitins. 

Agglutinins. — The  blood-serum  of  human  beings  as  well 
as  of  animals  suffering  from  certain  diseases  has  the  power 
of  causing  the  bacteria  of  the  disease  from  which  the  individual 
has  recovered  to  clump  into  larger  or  smaller  masses  in  liquid 
cultures  to  which  the  serum  is  added.  The  same  phenomenon 
is  observed  in  the  serum  of  animals  injected  with  repeated 
doses  of  cultures.  This  is  due  to  certain  substances  called 
agglutinins.  The  reaction,  while  it  is  more  or  less  specific, 
is  not  as  strictly  so  as  was  formerly  thought,  for  it  has  been 
found  that  a  given  agglutinin  may  cause  clumping  of  a  group 
of  nearly  related  bacteria;  such  an  agglutinin  is  called  a  group 
agglutinin;  and,  moreover,  under  certain  circumstances  the 

* Journal  American  Medical  Association.     September  3.  1904. 
tBesredka.     Bull,  de  VInst.  Past.  No.  13,  Vol.  IV.,  July  15,  1906.     p.  587. 


BACTERIAL    POISONS.  191 

bacteria  fail  to  clump  in  the  blood  of  patients  suffering  from  a 
given  disease.  Again,  in  some  cases  the  serum  in  a  certain 
disease  will  clump  bacteria  that  are  not  concerned  in  the 
production  of  the  disease.  Even  normal  serum  will  sometimes 
clump  bacteria.  The  serum  from  a  given  disease  is  said  to  be 
homologous  with  the  bacteria  causing  the  disease  and  heter- 
ologous  from  other  bacteria,  and  the  bacteria  are  said  to 
clump  or  not  to  clump  with  homologous  sera  as  the  case  may 
be.  Bacteria  are  also  homologous  or  heterologous  in  the  same 
sense  toward  sera. 

Park  and  Collins,*  in  summing  up  their  experience  in  the 
study  of  agglutinins  among  other  things,  state  that  the  injec- 
tion of  animals  with  bacteria  causes  the  production  of  agglu- 
tinins which  are  specific  for  the  organism  used  for  injection, 
but  in  addition  to  this  it  also  causes  the  production  of  agglu- 
tinins which  agglutinate  bacteria  other  than  those  injected;  not 
only  closely  allied  bacteria,  but  those  which  differ  widely  in  their 
biochemical  as  well  as  in  their  specific  agglutinating  characteris- 
tics. The  injection  therefore  forms  specific,  group  and  heterolo- 
gous agglutinins.  Of  these  there  is  at  first  more  specific  agglu- 
tinin  than  of  the  others.  Further  injection  may  cause  a  more 
or  less  rapid  diminution  of  the  specific  agglutinin  along  with 
a  less  rapid  diminution  of  the  others,  so  that  the  relative 
amounts  of  the  different  agglutinins  may  vary.  The  heter- 
ologous agglutinins  may  be  absorbed  by  injecting  the  immune 
animal  with  various  bacteria  other  than  the  one  used  for  the 
immunization  so  as  to  free  the  serum  from  all  but  the  specific 
agglutinin  so  that  it  will  clump  only  the  bacteria  which  were 
first  used  for  the  immunization.  Group  agglutinins  may  also 
be  absorbed  by  injecting  the  immune  animal  with  bacteria 
nearly  allied  to  that  used  for  immunization.  Thus  the  typhoid 
bacillus  will  absorb  many  group  glutinins  for  the  colon  bacil- 
lus. The  injection  of  bouillon  into  rabbits  will  frequently 

Journ.  Med.  Research.     XII.,  pp.  491-507.     1904. 


IQ2  MANUAL    OF    BACTERIOLOGY. 

produce  agglutinins  for  several  colon  types;  this  is  probably 
due  to  products  of  bacterial  growth  which  has  taken  place  in 
the  meat  extract  before  its  preparation  as  a  culture-medium. 
Agglutinins  and  lysins  bear  no  relation  to  one  another. 

Park*  points  out  that  the  majority  of  bacteria  do  not 
develop  sufficient  agglutinin  in  the  course  of  the  disease  which 
they  cause  to  be  detected,  as  in  the  case  of  tubercle,  influenza 
and  diphtheria  bacilli.  In  regard  to  group  agglutinins  he 
shows  that  bacteria  may  produce  such  a  large  amount  of  agglu- 
tinin in  the  course  of  the  infection  that  they  not  only  aggluti- 
nate themselves  with  the  blood  serum  from  the  patient,  but 
that  widely  different  bacteria  may  also  agglutinate  with  the 
serum.  Thus  he  found  that  an  animal  injected  with  staphy- 
lococcus  agglutinated  the  typhoid  fever  bacillus  in  the  propor- 
tion of  1-160  while,  before  the  serum  from  this  animal  agglu- 
tinated the  typhoid  bacillus  in  the  proportion  of  only  i-io. 
Park  gives  further  examples  of  the  same  sort.  But  on  the 
other  hand,  he  points  out  that  in  practice  such  conditions  will 
not  be  met  with,  and  it  may  be  regarded  as  certain  if  the 
typhoid  reaction  is  obtained  with  the  serum  from  a  patient 
in  the  proportion  of  1-50  in  two  hours  at  room  temperature 
that  the  patient  is  suffering  from  infection  with  a  member  of 
the  typhoid-colon  group  is  present,  probably  the  typhoid  bacillus. 

Parkf  also  found  that  bacteria  cultivated  on  homologous 
serai  lose  their  property  of  agglutination  with  the  kind  of 
serum  used  as  a  culture  medium,  but  recover  this  when  cul- 
tivated upon  the  ordinary  culture-media.  Weil§  obtained  a 
culture  of  typhoid  bacillus  from  an  abscess  in  the  thyroid  of  a 
typhoid  convalescent  which  did  not  agglutinate  with  the 
patient's  serum  nor  with  other  homologous  sera. 

*  Park.     Journ.    Infectious   Diseases.     Supplement  No.   2,   1906.     pp.   1—9. 

fW.  H.  Park.  Proceedings  of  the  New  York  Pathological  Society.  Vol.  IV., 
Nos.  i  and  2.  February  and  March,  1905. 

i  See  p.  191. 

§Weil.  Proceedings  of  the  New  York  Pathological  Society.  Vol.  IV., 
Nos.  i  and  2.  February  and  March,  1905. 


BACTERIAL    POISONS.  193 

In  spite  of  the  fact  that  agglutination  has  been  found  to  take 
place  spontaneously  in  cultures,  it  is,  nevertheless,  in  the  main 
a  specific  reaction,  and  is  employed  as  an  aid  in  the  diagnosis 
of  typhoid  fever,  where  it  is  spoken  of  as  the  Widal  or  Gruber- 
Wiclal  test.  Under  proper  precaution  it  is  valuable  in  this 
special  case,  and  will  be  referred  to  again  in  connection  with 
the  description  of  the  typhoid  bacillus. 

Other  bacteria  which  agglutinate  with  the  homologous 
sera  are:  Spirillum  of  cholera,  B.  pyocyaneus,  B.  proteus,  B. 
coli  communis,  Micrococcus  melitensis,  B.  mallei,  B.  tuberculo- 
sis, Diplococcus  pneumoniae,  B.  pestis  bubonicae,  and  B. 
dysenteriae.  Trypanosomes  also  agglutinate  with  homologous 
sera. 

Lysins. — There  are  certain  substances  found  normally 
present  or  produced  artifically  in  the  blood  which  have  the 
property  of  breaking  up  foreign  red  blood-cells  introduced 
into  the  circulation  or  into  the  blood-serum  outside  the  body. 
This  is  not  only  true  of  red  blood-cells,  but  certain  bacteria 
also  become  broken  up  when  introduced  into  the  blood  of 
certain  animals.  This  process  is  spoken  of  as  cytolysis,  and 
when  occurring  in  red  blood-cells,  is  called  hemolysis;  in 
bacteria,  bacteriolysis.  The  substances  causing  cytolysis  are 
called  lysins.  Lysins  have  also  been  obtained  from  cultures 
of  bacteria.  Thus  Besredka*  obtained  a  cytolysin,  strcpto- 
lysin  by  filtering  virulent  cultures  of  streptococcus  strongly 
hemolytic  for  the  red  corpuscles  of  many  animals.  Reudigerf 
corroborated  Besredka's  work  and  extended  his  observations 
upon  the  nature  of  streptolysin.  As  already  stated,  lysins 
for  certain  foreign  cells  are  normally  present  in  the  serum  of  cer- 
tain animals;  thus,  human  red  blood-cells  are  disintegrated  by 
sheep's  serum,  rabbit's  blood-serum  disintegrates  anthrax  bacilli, 
andnumerous  other  examples  exist  of  lysin  normally  present  in 

*  Ann.  de  PInst.  Past.  1901,  No.  15,  p.  880. 

f  Jour.  In].  Dis.  Vol.  IV.,  No.  2,  April  10,  1907.  pp.  277-281. 

13 


IQ4  MANUAL    OF    BACTERIOLOGY. 

blood-serum.  But  whether  normally  present  or  not,  specific 
lysins,  like  specific  agglutinins,  are  made  to  appear  in  the  blood- 
serum  of  animals  by  injecting  these  with  several  doses  of  sus- 
pensions of  cells.  Hemolysin  results  from  injecting  an  animal 
with  the  red  blood-cells  from  another  animal,  bacteriolysin  from 
the  injection  of  bacteria.  The  serum  of  the  blood  of  animals 
injected  in  this  way  is  called  immune  serum.  Thus  an  animal  in- 
jected with  B.  typholococcus  furnishes  typhoid-immune  serum; 
an  animal  injected  with  rabbit's  blood-corpuscles  furnishes 
rabbit-corpuscle-immune  serum.  Hemolysins  and  bacterio- 
lysins  are  quite  sharply  specific.  A  rabbit  injected  with  a 
suspension  of  red  corpuscles  from  the  blood  of  a  guinea-pig 
furnishes  hemolysins  which  destroys  guinea-pigs'  red  cells. 
A  guinea-pig  injected  into  the  peritoneal  cavity  with  repeated 
small  doses  of  the  cholera  spirillum  furnishes  a  peritoneal 
fluid  containing  a  bacteriolysin  specific  for  the  cholera  spirillum. 
Still,  group  lysins,  like  group  agglutinins,  are  also  found,  for 
while  lysis  takes  place  more  promptly  and  in  smaller  amounts 
with  the  cells  of  the  same  species  of  animal  or  with  the  same 
kind  of  bacteria  with  which  the  animal  furnishing  the  cytolytic 
serum  has  been  injected,  it  also  occurs  in  a  less  marked  degree 
with  cells  from  nearly  related  animals  or  with  nearly  similar 
bacteria.* 

The  same  serum  is  not  equally  potent  for  different  bacteria, 
and  the  serum  from  different  animals  of  the  same  or  of  different 
species  varies  in  bactericidal  potency  for  the  same  organism. 
The  chemical  reaction  of  the  serum  seems  to  exert  some  effect 
upon  this  quality  of  the  serum.  The  more  alkaline  the  serum 
the  more  -potent  is  its  action  apparently,  and  consequently 
the  venous  blood  has  been  found  sometimes  to  furnish  more 
potent  serum  than  the  arterial  blood  from  the  same  animal. 

Serum  loses  in  bactericidal  potency  on  standing  after  being 

*Further  reference:  Pribram.  Kolle  and  Wassermann.  Erganzungsband. 
1906.  pp.  291-346. 


BACTERIAL   POISONS.  IQ5 

drawn  from  the  animal;  and  the  higher  the  temperature  to 
which  the  serum  is  exposed  the  more  rapid  the  loss  of  potency. 
It  may  remain  potent  for  several  days  or  for  even  a  week  or 
more  in  the  refrigerator,  but  if  kept  at  body  temperature 
it  generally  loses  all  bactericidal  properties  in  three  or  four 
hours.  Heating  at  55°  or  56°  C.  for  ten  or  fifteen  minutes  also 
robs  the  serum  of  its  bactericidal  power,  or  rather  this  treat- 
ment of  immune  serum  suspends  its  bactericidal  power,  which 
is  restored  by  the  addition  of  a  small  amount  of  fresh  serum. 
This  suspension  of  bactericidal  power  by  heating  at  55°  or 
56°  C.  is  called  inactivating  the  serum.  Inactivated  serum 
to  which  fresh  serum  is  added,  and  which  has  had  its  bacterio- 
lytic  properties  restored  in  this  way,  is  termed  reactivated 
serum.  There  are  probably  many  circumstances  which  in- 
fluence the  bacteriolytic  property  of  the  serum.  The  chemi- 
cal reaction  of  the  serum  has  been  found  to  influence  the  bac- 
tericidal power,  and  doubtless  there  are  other  as  yet  obscure 
circumstances  which  raise  or  lower  this  power  of  the  serum. 
The  nature  of  bacteriolysins  will  be  found  discussed  at  some 
length  below,  and  it  would  not  seem  at  all  improbable  that 
there  may  be  more  of  these  at  one  time  than  at  another  present 
in  the  blood-serum.  Indeed,  the  production  of  bacteriolysis 
in  serum  of  the  living  animal  seems  to  be  easily  influenced  one 
way  or  another,  and  it  would  not  be  unreasonable  to  regard 
them  as  varying  from  time  to  time  under  even  slightly  chang- 
ing conditions  of  the  body. 

Trommsdorf*  noticed  that  human  sera  derived  from  normal 
individuals  as  well  as  from  those  suffering  from  various  dis- 
eases vary  greatly  in  bacteriolytic  power.  Pettersonf  found 
the  same  thing  with  chickens. 

*Richard  Trommsdorf.  Ueberden  Alexingchaltnovmaler  und  pathologischer 
mauschlicher  Blutsera.  Centralbl.  /.  Bakt.,  etc.  abt  i.  Orig.  Bd.  32,  No.  6, 
pp.  436-449.  1902. 

fj.  Morgenroth  and  H.  Sachs.  Ueber  die  Comp'etierbarkeit  der  ambocep- 
toren.  Berl.  klin.  Woch.  Jahrg.  39,  No.  27,  pp.  631-633. 


196  MANUAL    OF    BACTERIOLOGY. 

Morgenroth  and  Sachs*  found  great  variation  in  cytolytic 
power  in  serum  of  various  sorts.  Thus  the  serum  fiom  a  horse 
at  one  drawing  was  hemolytic  for  rabbits'  corpuscles  but  not 
for  those  of  guinea-pigs;  three  days  later  the  serum  from  the 
same  horse  was  strongly  hemolytic  for  guinea-pigs'  corpuscles, 
but  only  very  slightly  for  rabbits'  corpuscles;  twenty-three  days 
later  the  serum  from  this  horse  was  not  hemolytic  for  guinea- 
pigs'  corpuscles,  but  strongly  hemolytic  for  rabbits'  corpuscles. 

It  is  therefore  evident  that  the  cytolytic  power  of  serum  is 
very  variable.  Not  only  does  the  blood  from  different  indi- 
viduals of  the  same  species  differ  in  this  respect,  but  the  serum 
from  the  same  individual  differs  from  time  to  time.  This  is 
probably  the  case  with  all  animals. 

The  peculiar  behavior  of  immune  sera  on  dilution  will  be 
described  under  immunity. 

Precipitins. — Precipitins  are  bodies  which  develop  in  the 
serum  of  animals  which  have  been  given  subcutaneous  injec- 
tions of  albuminous  substances,  and  which,  added  to  solutions 
of  the  albumin  with  which  the  animals  have  been  injected, 
cause  these  to  become  cloudy  and  finally  form  a  precipitate. 
Thus  a  rabbit's  blood-serum  may  be  sensitized  by  injections 
of  hen's  egg-albumen,  and  the  rabbit's  blood-serum  will  then 
precipitate  hen's  egg-albumen.  It  may,  however,  imperfectly 
precipitate  albumen  from  the  egg  of  a  species  closely  allied 
to  the  hen. 

Again,  a  rabbit  injected  a  few  times  at  intervals  of  a  day 
or  two  with  human  blood-serum  furnishes  serum  which  even 
in  small  quantities  causes  precipitation  even  in  a  weak  solution 
of  human  blood-serum,  such  as  may  be  obtained  from  old 
dried  blood-spots. 

The  effect  produced  by  the  injection  of   foreign  albumin 


*  Alfred  Petterson.  Ueber  die  natiirliche  milzbrandimmunitatdeshundesund 
des  huhns.  Cent.  }.  bakt.,  abt.  i,  Orig.,  bd.  33,  no.  8,  p.  613-626.  Jena, 
Apr.  4,  1903. 


BACTERIAL    POISONS.  IQ7 

is  regarded  as  analogous  to  that  produced  by  the  injection  of 
bacteria,  and  consequently  the  serum  of  the  animal  injected 
with  albumin  is  spoken  of  as  immune  serum  in  the  same  way 
as  the  serum  from  an  animal  injected  with  bacteria  is  so  desig- 
nated. The  serum  from  the  rabbit  in  the  last  example  is 
human  serum  immune-rabbit-serum. 

The  term  homologous  is  employed  to  denote  the  relation 
between  the  immune  serum  and  the  albumin  used  in  its  pro- 
duction. Thus,  in  the  example  just  given,  the  rabbit's  serum 
and  human  serum  would  be  called  homologous  the  one  with 
the  other. 

The  reaction  is  very  sensitive,  the  immune  serum  causing 
clouding  in  solutions  containing  very  small  traces  of  the  homol- 
ogous serum.  Rabbits  are  usually  employed  for  the  production 
of  immune  sera  for  the  precipitation  reaction,  and  seem 
specially  adapted  to  the  purpose. 

The  reaction  is  of  great  value  in  determining  the  kind  of 
blood  in  any  doubtful  case,  and  is  applied  practically  in  foren- 
sic medicine  to  determine  the  character  of  suspicious  blood- 
stains. 

In  the  precipitin  reaction,  as  this  is  called,  there  is  group 
precipitation,  it  is  true.  Thus,  human  and  monkey's  sera 
react  with  the  same  precipitin  and  dog's  and  wolf's  sera 
respond  to  the  same  precipitin.  Bacterial  precipitins  have 
also  been  obtained  by  injecting  animals  with  bacteria.  In 
this  case  nitrates  obtained  by  filtering  bacterial  cultures  or 
suspensions  through  porcelain  filters  give  a  cloud,  with  ulti- 
mate precipitation,  when  treated  with  a  drop  of  homologous 
serum.* 

*Norris.  The  Bacterial  Precipitins.  Journal  of  Injections  Diseases.  Vol. 
T.,  p.  463.  1904 


CHAPTER  VI. 
IMMUNITY. 

Studies  in  immunity  have  led  to  remarkably  uniform  results 
in  so  far  as  the  facts  are  concerned  and  there  is  great  unanimity 
in  regard  to  the  actual  observations,  both  of  the  processes 
which  take  place  spontaneously  in  nature,  as  well  as  of  those 
which  follow  in  the  intentional  experiments.  There  is,  how- 
ever, great  difference  of  opinion  upon  the  interpretation  of  these 
phenomena,  and  several  opposing  theories  have  been  advanced 
in  regard  to  the  mechanism  concerned,  each  theory  finding  very 
eminent  supporters. 

In  view  of  these  facts,  it  is  necessary,  in  discussing  immu- 
nity, to  give  a  definition  covering  its  broadened  application,  to 
cite  the  observations  which  have  been  recorded  and  to  present 
the  prevalent  explanations  offered  by  the  various  authorities. 

It  is  scarcely  necessary  to  go  into  a  discussion  of  the  various 
theories  which  have  been  advanced  from  time  to  time,  but  which 
have  now  been  abandoned. 

Immunity,  as  formerly  studied,  embraced  only  considerations 
of  the  insusceptibility  of  individuals  or  of  races  to  an  attack 
of  a  given  infectious  disease.  But  the  modern  conception 
is  broader  than  this,  and  it  is  no  bnger  confined  to  immunity 
proper,  but  extends  to  certain  other  processes  which  have  been 
found  to  bear  a  close  resemblance,  in  certain  respects,  to  im- 
munity, and  to  be  governed  by  laws  very  similar  to  those  which 
govern  the  latter. 

Immunity  at  present  is  made  to  include,  besides  insuscepti- 
bility to  infection — i.  e.,  resistance  to  the  inv-asion  of  living 
bacteria — the  processes  concerned  in  the  forming  of  the  anti- 

'198 


IMMUNITY.  199 

bodies,  antitoxins  proper,  antiagglutinins,  antilysins  and  anti- 
precipitins. 

Resistance  to  Infection. — Immunity  from  infectious  dis- 
eases is  either  natural  or  acquired,  active  or  passive. 

By  natural  immunity  is  meant  the  inherited  power  possessed 
by  certain  races  or  individuals,  independently  of  size,  habits 
or  surroundings,  to  resist  infection  to  which  other  races  or  in- 
dividuals are  subject.  This  is  illustrated  by  many  examples. 
Rats  are  ordinarily  insusceptible  to  anthrax,  whereas  mice, 
guinea-pigs,  sheep,  cattle — in  short,  most  animals — are  very 
susceptible.  Mice  are  not  susceptible  to  diphtheria  poison  on 
inoculation,  while  horses,  sheep,  goats,  guinea-pigs  and  many 
other  animals  are  very  susceptible.  Even  very  nearly  related 
species  or  varieties  often  show  difference  in  susceptibility. 
House-mice  are  susceptible  to  mouse  septicemia,  European 
field-mice  are  not.  On  the  other  hand,  with  glanders,  house- 
mice  and  white  mice  are  less  susceptible  than  European  field- 
mice  are  to  this  disease.  Negroes  are  insusceptible  to  certain 
diseases  to  which  the  white  race  is  very  subject,  and  also  the 
reverse. 

Instances  of  individual  immunity  are  seen  in  every  epidemic, 
where  persons  escape  when  they  are  under  the  same  conditions 
as  those  who  have  contracted  the  disease.  Instances  also  occur 
in  which  nurses  and  others  thrown  with  cases  of  highly  infec- 
tious diseases  escape.  Some  of  these  cases,-  it  is  true,  belong 
more  properly  to  the  category  of  immunity  acquired  by  recovery 
from  an  attack,  since  nurses  and  others  thrown  in  contact  with 
an  infectious  case  may  suffer  a  very  mild  attack  or  even,  prob- 
ably become  immunized  without  showing  any  symptoms  of 
disease.  Nurses  and  physicians  have  been  found  with  diph- 
theria bacilli  in  their  throats  and  yet  not  showing  any  symptoms. 
Of  course,  these  persons  may  have  had  natural  immunity,  but 
it  is  equally  possible  that  they  may  have  become  gradually 
immunized. 


200  MANUAL   OF    BACTERIOLOGY. 

Furthermore,  as  just  stated,  immunity  probably  also  results 
from  intimate  contact  with  diseased  individuals. 

Acquired  immunity  follows  recovery  from  a  spontaneous 
attack  of  certain  diseases,  and  it  also  results  from  intentional 
inoculation.  Immunity  after  recovery  is  so  familiar  that  no 
illustration  is  necessary.  But  not  every  infectious  disease 
leaves  immunity  behind;  many,  on  the  contrary,  are  followed 
by  no  increased  resistance,  or  if  there  is  increased  resistance, 
it  is  very  transitory;  some  even  tend  to  increase  susceptibility. 
Examples  of  infectious  diseases  leaving  no  lasting  immunity, 
and  even  in  some  cases  apparent  increased  susceptibility,  are 
erysipelas,  diphtheria,  influenza,  pneumonia,  gonorrhea. 
In  those  cases  in  which  immunity  follows  recovery  from  a 
spontaneous  attack  of  an  infectious  disease  the  process  is 
probably  the  same  as  that  which  takes  place  after  intentional 
inoculation. 

Immunity  acquired  after  intentional  inoculation  is  produced 
either  by  inoculation  with  material  which  produces  a  mild 
type  of  disease,  as  in  vaccination  for  small-pox;  or  by  giving 
at  short  intervals  inoculations  of  the  disease-producing  virus 
of  graded  strength,  beginning  with  greatly  attenuated  material, 
and  followed  with  stronger  and  stronger  material,  as  in  inocu- 
lation for  hydrophobia  and  anthrax;  or  by  injections  of  larger 
and  larger  amounts  of  bacterial  poison,  as  in  the  production 
of  antitoxin  for  diphtheria  and  tetanus;  or,  finally,  by  the  in- 
jection of  the  antitoxin  formed  in  the  blood  by  the  method 
last  mentioned,  as  in  the  treatment  of  diphtheria  and  tetanus, 
for  this,  after  all,  is,  in  a  way,  a  process  of  immunization. 

Small-pox  and  Vaccination. — The  origin  of  vaccination 
against  small-pox  with  the  virus  of  cow-pox  has  been  de- 
scribed in  the  historical  sketch.  The  nature  of  the  protection 
furnished  by  this  virus  has  been  the  subject  of  much  contro- 
versy. The  opinion  of  the  present  day  inclines  to  regarding 
vaccinia  as  small-pox  which  has  been  modified  by  passage 


IMMUNITY.  201 

through  a  relatively  insusceptible  animal.  Councilmann* 
and  his  co-workers  regard  cow-pox  and  small-pox  as  identical. 
But  Law,  Salmon  and  Smith  f  regard  the  two  diseases  as 
different. 

This  question  cannot  be  settled  with  certainty  until  the  or- 
ganisms causing  small-pox  and  vaccinia  have  been  isolated 
in  pure  culture.  Their  identity  and  mode  of  action  may  then 
be  determined. 

Small-pox  has  been  inoculated  into  calves  and  passed 
through  other  calves  in  succession,  producing  finally  an  erup- 
tion indistinguishable  from  cow-pox.  Not  only  does  recovery 
from  spontaneous  or  artificial  cow-pox  protect  against  small- 
pox, but  it  has  been  shown  that  recovery  from  small-pox 
protects  against  cow-pox. 

Attenuated  Virus  and  Cultures  in  Which  the  Bacteria 
are  Killed. — Pasteur  conceived  the  idea  of  attenuating  the 
virulence  of  the  bacilli  of  fowl-cholera  by  prolonged  exposure 
to  the  air.  He  made  use  of  the  virus  thus  attenuated  as  a 
vaccine  against  the  disease. 

A  nearly  similar  principle  was  shortly  afterward  applied  by 
him  to  the  preparation  of  a  vaccine  against  anthrax.  When 
anthrax  bacilli  were  cultivated  at  a  temperature  of  43°  C., 
Pasteur  obtained  bacilli  of  very  slight  virulence.  Such  bacilli 
did  not  produce  death  when  inoculated  into  animals  that  were 
ordinarily  susceptible.  Yet  animals  that  were  vaccinated  with 
this  virus  were  able  afterward  to  resist  inoculation  with  fully 
virulent  anthrax  bacilli.  (See  Bacillus  anthracis,  Part,  IVj. 

Successful  methods  of  inoculation  have  been  found  for 
rinderpest,  an  infectious  disease  of  cattle  in  which  the  digestive 
organs  are  mainly  involved.  There  are  three  ways  which  are 
eflficacious:J  (i)  Injection  of  bile  from  an  animal  recently 
dead  of  rinderpest;  (2)  inoculation  of  glycerinated  bile,  fol- 

*  Journ.  Med.  Research.     VII.     No.  i. 

^Diseases  of  Cattle.     Dept.  Agr.  Pub.  1904.     p.  237  and  p.  426. 

^Diseases  of    Cattle.     Dept.    Agricult.     Publication.     1904.     pp.    380-381. 


202  MANUAL    OF    BACTER  OLOGY. 

lowed  by  injection  of  pure  bile  or  virulent  blood;  (3)  by  the 
simultaneous  injection  of  strong  standarized  serum  and  viru- 
lent blood. 

In  the  case- of  erysipelas  of  swine  (French,  rouget;  German, 
Schweinerothlauf)  Pasteur  secured  bacilli  of  diminished  viru- 
lence by  injecting  virulent  bacilli  into  relatively  insusceptible 
animals.  The  animal  used  was  the  rabbit.  The  bacilli  were 
passed  through  several  rabbits  in  succession.  Cultures  taken 
from  the  last  of  the  series  produced  a  milder  form  of  the  disease 
and  amount  of  immunity  to  a  certain  degree.  The  practical 
value  of  this  inoculation  is  not  settled. 

In  still  another  disease,  black-leg  of  cattle  or  symptomatic 
anthrax  (French,  charbon  symptomatique;  German,  Rausch- 
brand),  an  attenuated  vims  is  secured  by  the  use  of  heat. 
The  pulp  from  the  infected  muscle  of  a  diseased  animal, 
containing  the  bacilli,  is  squeezed  from  it  and  heated  to  a 
temperature  of  95°  to  99°  C.  for  six  hours.  The  dried  material 
mixed  with  water  constitutes  the  vaccine.  The  Depart- 
ment of  Agriculture  of  the  United  States  now  furnishes  this 
vaccine  free  to  farmers.  The  results  of  this  method  are  said 
to  be  very  satisfactory.*  In  the  human  disease,  bubonic  plague, 
a  nearly  similar  procedure  has  been  proposed  by  Haffkine. 
To  protect  against  plague,  cultures  of  plague  bacilli  are  used 
which  have  been  previously  sterilized  by  heat,  with  carbolic 
acid  added  as  a  preservative.  (See  section  on  Bubonic  Plague, 
Part  IV.)  In  the  preparation  of  these  " vaccines"  it  is  of  the 
utmost  importance  to  use  pure  cultures,  and  to  be  sure  that 
the  cultures  are  dead.  It  is  not  enough  to  subject  them  to 
heating  or  to  the  action  of  other  germicidal  agents  as  recom- 
mended in  books,  but  they  should  in  all  cases  be  tested  with 
cultures  to  determine  whether  all  the  bacteria  have  been  cer- 
tainly killed. 

*See  Annual  Reports,  Bureau  of  Animal  Industry,  U.  S.  Department  of  Agri- 
culture. 


IMMUNITY.  203 

Inoculation   Against   Rabies    or  Hydrophobia.* — The 

immunity  produced  in  this  case  probably  depends  upon  prin- 
ciples similar  to  those  underlying  the  examples  related  on  the 
preceding  pages.  But  this  question  cannot  be  regarded  as 
settled  until  the  organism  of  rabies  has  been  isolated  and  cul- 
tivated. Attempts  to  discover  this  organism  have,  as  yet,  been 
futile,  though  certain  minute  bodies,  bodies  of  Negri,  have  been 
observed  within  ganglion-cells  of  the  central  nervous  system 
from  cases  of  rabies,  and  it  has  been  claimed  that  they  are  pro- 
tozoa and  the  cause  of  the  disease.  Whether  this  is  true  or  not, 
Negri  bodies  make  a  most  valuable  means  of  rapid  diagnosis. 

Frothinghamf  regards  the  presence  of  the  Negri  bodies  as 
sufficient  for  diagnostic  purposes  without  animal  inoculation. 
If  these  are  not  found  in  smear  preparations  from  Ammon's 
horn,  they  must  be  further  sought  in  sections  from  this  region 
or  in  the  cerebellum  or  in  the  Gasserian  ganglion.  If  all  these 
tests  are  negative,  and  there  are  no  lesions  in  the  Gasserian 
ganglion,  then  animal  inoculation  may  be  resorted  to  merely 
to  allay  uneasines  of  the  patient. 

DavisJ  calls  attention  to  the  fact  that  the  Negri  bodies  are 
sometimes  absent  in  undoubted  cases  of  hydrophobia.  Such 
cases  are  very  few,  and  these  may  be  diminished  by  improved 
technic.  For  fixing  Zenker's  fluid  and  corrosive  sublimate 
are  available,  formalin  is  not.  No  special  stain  is  required; 
any  ordinary  nuclear  stain  will  answer  as  well  as  Koch,  Unna 
or  other  modifications  of  Romanowsky.  Davis  advises  that 
the  suspected  animal  should  be  kept  alive  in  a  cage  for  obser- 
vation. If  it  has  hydrophobia  it  will  die  in  a  few  days.  The 
brain  should  be  removed  after  death  and  examined  for  Negri 
bodies  and  if  these  are  found  any  person  who  has  been  bitten 
should  be  given  the  Pasteur  treatment.  If  the  bodies  are  not 

*  For  a  review  of  works  on  rabies  see  Remlinger.  Bulletin  de  I'lnstitut  Pasteur. 
II.,  Nos.  19  and  20.  1904. 

f  Journ  Med.  Research.     XIV.  1905.     pp.  471-489. 
Uourn  A.  M.  A.     July  14,  1906.     pp.  87-89. 


204  MANUAL    OF    BACTERIOLOGY. 

found  -  animals  should  be  inoculated.  He  makes  the  remark 
that  whatever  the  nature  of  these  bodies,  whether  they  are 
parasites  or  degenerated  cells,  they  are  characteristic  of  hy- 
drophobia, but  are  not  identical  with  the  virus.  For  the 
hypocampus  where  they  are  found  in''greatest  abundance  is  not 
more  virulent  than  other  parts  of  the  brain  in  which  they  may 
not  be  found.  The  stain  when  used  is  diluted  in  the  proportion 
of  one  drop  of  the  stain  to  i  c.c.  of  distilled  water  which  has 
been  rendered  alkaline  by  the  addition  of  one  drop  of  a  i  per 
cent,  solution  of  potassium  carbonate  to  10  c.c.  of  water.  This 
diluted  stain  is  poured  over  the  dried  specimen  and  allowed  to 
remain  for  from  one-half  to  three  hours  or  even  longer.  The 
preparation  is  then  washed  in  running  tap  water  for  one  to 
three  minutes,  and  dried  with  filter-paper.  The  cytoplasm  of  the 
Negri  bodies  takes  a  blue  color,  the  central  bacillus  and  chro- 
matoid  granules  stain  blue-red  or  azure.  The  tinge  depends 
upon  the  thickness  of  the  specimen.  The  cytoplasm  of  the 
nerve-cells  stains  blue  also,  but  the  nuclei  of  the  nerve-cells 
stain  red,  the  nucleoli  dull  blue. 

For  diagnostic  purposes  the  method  may  be  shortened  as 
follows:  Methyl  alcohol,  five  minutes;  equal  parts  of  the 
Giemsa  solution  and  distilled  water,  ten  minutes. 

In  Mallory's  method  the  smears  are  fixed  in  Zenker's  fluid 
for  one-half  hour;  rinsed  in  tap  water;  one-quarter  hour,  95  per 
cent,  alcohol  and  iodin;  95  per  cent,  alcohol,  one-half  hour; 
absolute  alcohol,  one-half  hour;  eosin  solution  twenty  min- 
utes; rinsed  in  tap  water;  methyline  blue  solution,  fifteen 
minutes;  95  per  cent,  alcohol,  one  to  five  minutes;  dry  with 
filter-paper. 

Anna  Wessel  Williams  and  Lowden*  recommend  the  use  of  smear  preparations 
of  the  brain  for  examination  of  the  Negri  bodies  for  the  reason  that  the  bodies  ap- 
pear more  characteristic  in  such  preparations  than  in  preparations  made  in  other 
ways.  The  smears  are  prepared  as  follows: 

*Williams  and  Lowden.     Journ.  Infectious  Diseases.     Vol.  III.,  1906.     pp 
452-483. 


IMMUNITY.  205 

Very  thin  slices  one  each  from  near  the  fissure  of  Rolando,  from  Ammon's 
horn,  and  from  the  cerebellum  are  cut  at  right  angles  to  the  surface  taking  the 
gray  matter.  These  are  spread  upon  scrupously  clean  slides,  and  rubbed  over 
in  one  direction  gently  with  cover-glasses.  The  smears  are  allowed  to  dry  in  the 
air,  and  stained  by  Giemsa's  or  by  Mallory's  methods.  Giemsa's  stain 
as  last  recommended  consists  of:  Azure  II  0.8;  eosin  3.0  gr.;  glycerin 
(chem.  pure)  250  c.c.;  methyl  alcohol  (chem.  pure)  250  c.c.  Both  glycerin 
and  alcohol  are  heated  at  60°  C.  The  dyes  are  put  into  the  alcohol  and  the 
glycerin  is  added  slowly,  stirring.  The  mixture  is  allowed  to  stand  at  room 
temperature  over  night,  and  is  ready  for  use  after  filtering. 

Luzzani*  reports  the  results  of  himself  and  those  of  others 
in  regard  to  the  presence  of  Negri  bodies.  This  report  shows 
that  the  bodies  were  not  found  in  9  out  of  296  cases  in  which 
hydrophobia  diagnosis  was  confirmed  by  inoculation.  In  the 
other  cases  it  was  found  either  in  sections  or  smear  preparations. 

Marie, f  contrary  to  the  usual  experience,  found  that  the  blood 
of  animals  affected  with  rabies  may  be  virulent  under  cir- 
cumstances not  yet  determined. 

Di  Vestea  J  found  that  the  virus  of  rabies,  which  is  well  pre- 
served by  glycerin  in  the  nerve-tissue,  is  not  so  preserved  when 
filtered  through  unglazed  porcelain.  In  fact,  the  virus  after 
nitration  is  in  all  respects  much  more  labile  than  when  not 
filtered.  Di  Vestea  comes  to  the  conclusion  from  this  ob- 
servation that  only  the  young  forms  of  the  parasite  pass 
through  the  filter.  Moreover,  that  the  results  with  glycerin 
as  a  preservative  are  in  favor  of  the  idea  that  rabies  virus  is 
intracellular,  probably  since  the  glycerin  kills  the  young  para- 
sites which  have  not  yet  entered  the  brain  cells,  but  not  those 
contained  in  the  brain  cells. 

Remlinger§  found  that  centrifugalizing  rabies  virus  for  one 

*Arch.  per  le  sc.  med.  T.  XXVIII.,  No.  34,  pp.  521-540.  Abstract  in  Bulle- 
tin de  I'lnst.  Pasteur.  Vol.  III.,  No.  7.  April  15,  1905.  pp.  297-298. 

fCompt.  Rendus  Soc.  Biologic,  T.  LVIII.  25  Mars,  1905,  p.  544.  Abstract 
in  Bui.  de  I'lnst.  Past.  Vol.  III.,  No.  n.  June  15,  1905.  p.  461. 

£Comm.  Ac.  Medic.  Pise,  24  Jan.,  1905.  p.  2.  Abst.  Bui.  de  I'lnst.  Pasteur. 
T.  III.,  No.  8.  30  Avril,  1905.  p.  338. 

§  Compt.  Rendus  Soc.  Biologic.  T.  LVIII.,  7  Janv.,  1905.  p.  27.  Abst. 
Med.  de  I'lnst.  Pasteur.  T.  III.,  No.  7.  15  Avril,  1905.  p.  301. 


206  MANUAL    OF    BACTERIOLOGY. 

hour  with  noo  revolutions  a  minute,  using  the  virus  diluted  i 
to  50  and  i  to  100  deprives  the  virus  of  its  virulence.  He  also 
found  that  the  virus  traveled  along  the  nerves  and  not  the 
lymphatics  in  the  rare  cases  of  successful  inoculation  of  ani- 
mals into  a  pathologically  hypertrophied  ganglion.* 

Tizzoni  and  Bongiovannif  found  that  radium  rays  not  only 
destroy  the  virulence  of  rabies  virus  in  vitro,  but  also  counter- 
act the  effects  of  the  virus  in  animals  inoculated  and  exposed  to 
the  rays,  and  furthermore  the  rays  transform  the  virus  into 
an  excellent  antirabic  virus.  Still  more  remarkable  is  their 
statement  that  radium  rays  cure  the  disease  after  symptoms 
have  developed. 

Pasteur  discovered  that  rabies  could  be  produced  in  animals 
by  inoculation  under  the  dura  mater  with  portions  of  the  spinal 
cord  of  a  dog  suffering  from  hydrophobia.  He  also  found  that 
successive  passages  through  a  series  of  rabbits  greatly  increase 
the  virulence  of  the  virus,  as  indicated  by  a  much  shorter  period 
of  incubation  after  inoculation.  The  first  rabbit  of  the  series 
inoculated  with  the  "street"  rabies  virus — i.  e.,  from  a  spon- 
taneous case  in  a  dog — dies  in  about  two  weeks,  and  each 
succeeding  rabbit  dies  in  a  shorter  and  shorter  time  until, 
ultimately  the  incubation  period  is  reduced  to  six  or  seven  days. 
Beyond  this  the  strength  of  the  virus  cannot  be  increased,  and 
is  called  "virus  fixe,"  or  the  fixed  virus.  Pasteur  found,  more- 
over, that  the  cord  of  the  rabbit  which  has  attained  this  degree 
of  virulence  is  attenuated  by  various  agencies,  notably  by  dry- 
ing, and  that  animals  injected  with  this  attenuated  virus  can 
withstand  inoculations  of  more  potent  virus.  By  drying  for 
various  lengths  of  time  a  series  of  "vaccines"  of  exactly  graded 
potency  is  obtained,  and  starting  with  the  vaccine  of  least  po- 
tency an  animal  can  be  inoculated  with  increasingly  potent  virus 
until  it  will  withstand  inoculations  of  the  virus  fixe  itself. 

*Bull.  de  I'lnst.  Past.     Vol.  IV.,  1906      p.  448. 

fAcad.  d.  sciences  de  Bologne.  Avril,  1905.  p.  6.  Abst.  Bui.  de  I'lnst. 
Pasteur.  T.  III.,  No.  13.  15  Juillet,  1905.  p.  582. 


IMMUNITY.  207 

Omitting  all  but  the  chief  details,  the  vaccines  against 
hydrophobia  are  prepared  as  follows:* 

The  cord  of  a  rabbit  dead  from  the  subdural  inoculation 
of  virus  fixe  is  hung  up  in  a  long  glass  cylinder  in  the  bottom 
of  which  is  placed  potassium  hydrate.  The  cylinder  is  placed 
in  a  cool  place,  and  every  day  small  bits  of  the  cord  are  cut 
off  and  preserved  in  a  vial  of  glycerin.  The  virus  which  has 
been  dried  for  thirteen  or  fourteen  days  is  no  longer  capable  of 
producing  hydrophobia  in  rabbits,  but  an  animal  inoculated 
with  it  can  withstand  inoculation  with  the  cord  dried  for  a 
shorter  time,  and  after  inoculation  with  the  latter  withstands 
inoculations  with  cord  dried  for  a  still  shorter  time. 

In  human  beings  it  is  customary  to  start  with  the  virus  which 
has  been  dried  for  nine  or  ten  days,  injecting  subcutaneously 
emulsions  of  the  dried  cord,  and,  if  time  permits,  to  give  an 
inoculation  every  day  with  virus  dried  for  a  shorter  and  shorter 
time.  As  the  incubation  period  for  human  beings  bitten  by  a 
mad  dog  is  quite  long, — about  six  or  eight  weeks, — there  is 
ample  time  to  run  in  all  the  inoculations  if  these  are  begun 
promptly,  and  if  in  this  way  the  individual  is  made  to  with- 
stand the  virus  fixe,  it  is  more  than  probable  that  the  weaker 
virus  from  the  dog  will  not  be  able  to  cause  any  disease. 

Where  much  time  has  elapsed  after  the  bite  of  the  mad 
dog,  it  is  sometimes  the  practice  to  give  three  or  more  injections 
of  increasing  strength  every  day. 

These  inoculations  against  hydrophobia  have  proved 
to  be  most  valuable,  as  the  large  number  of  reports  from  various 
Pasteur  institutes  in  various  parts  of  the  world  abundantly 
prove.  According  to  statistics,  collected  by  Ravenel,  based  on 
many  thousands  of  cases,  the  mortality  from  rabies  in  those 
so  treated  is  less  than  i  per  centj 

*  Valuable  information  in  regard  to  the  preparation  and  mode  of  using  hydro- 
phobia virus  was  contributed  in  a  personal  letter  to  Dr.  Williams  by  Dr.  Jas.  G. 
Cummings,  Pasteur  Institute,  University  of  Michigan. 

fSee  Ravenel  and  McCarthy.  University  of  Pennsylvania  Medical  Bulletin. 
June,  1901.  Also  editorial  in  Phildelphia  Medical  Journal.  March  14,  1903. 
And  Mohler.  Twelfth  Annual  Report  of  the  United  States  Bureau  of  Animal 
Industry. 


208  MANUAL    OF    BACTERIOLOGY. 

In  all  cases  where  a  human  being  has  been  bitten  by  a  dog 
that  is  suspected  of  having  hydrophobia,  the  individual  should 
submit  himself  to  the  Pasteur  treatment  as  soon  as  possible, 
if  it  is  feasible  to  do  so.  Since  this  treatment,  if  done  by  com- 
petent hands,  insures  the  person  who  has  been  bitten  against 
the  danger  of  the  development  of  the  disease. 

In  order  to  find  out  whether  the  dog  has  hydrophobia,  the 
animal  may  either  be  put  up  and  observed  to  see  whether  char- 
acteristic symptoms  develop,  or  it  may  be  immediately  killed. 
In  the  latter  case  the  brain  and  cord  should  be  examined  for 
the  presence  of  the  Negri  bodies  already  alluded  to,  and  the 
intervertebral  ganglia  for  the  presence  of  round-cell  infiltration 
which  is  often  marked.  Rabbits  or  guinea-pigs  should  also  be 
inoculated  under  the  dura.  As  stated  in  the  beginning,  it  is 
safer  not  to  wait  for  the  result  of  the  examination,  but  to  sub- 
ject the  person,  who  is  bitten,  to  the  Pasteur  treatment  in 
advance  of  this,  unless  it  is  improbable  that  the  dog  is  mad. 
The  examination  consists  in  looking  for  the  Negri  bodies  and 
in  subdural  inoculation  of  rabbits.  If  the  cord  of  the  dog  can 
be  obtained,  the  intervertebral  ganglia  will  show  round-cell 
infiltration.  All  other  ganglia  may  also  show  this  change. 
The  plexiform  and  Gasserian  ganglia  are  convenient  for 
examination. 

Great  care  must  be  taken  that  the  operator  may  not  acciden- 
tally infect  himself. 

Antitoxins. — Antitoxins  have  as  yet  been  produced  for  the 
extra-cellular  toxins  only,  and  only  those  diseases  which  are 
caused  by  bacteria  which  form  extra-cellular  toxins  have  been 
successfully  combated  in  this  way.  Antiendotoxins,  on  the 
other  hand,  have  not  yet  been  satisfactorily  produced.  Buxton* 
found  that  rabbits  immunized  with  typhoid  bacilli  do  not 
become  appreciably  more  resistant  than  normal  rabbits  to 
the  endotoxin  of  the  typhoid  bacilli. 

*  Journ.  Med.  Research,     V.  XVI.,  No.  2,  May,  1907.     p.  264. 


IMMUNITY.  209 

The  methods  used  in  the  production  of  antitoxins  were  intro- 
duced by  Behring,  who  found  that  by  injecting  susceptible 
animals  with  increasing  amounts  of  extra-cellular  toxin  he 
produced  in  the  blood-serum  of  the  injected  animal  certain 
changes  which  made  the  serum  capable  of  counteracting  the 
same  toxin  when  injected  into  other  animals.  Thus,  a  sheep 
treated  with  increasing  doses  of  diphtheria  toxin,  beginning 
with  very  small  amounts,  furnishes  blood-serum  which  protects 
other  sheep  or  guinea-pigs  or  other  susceptible  animals  from 
fatal  doses  of  diphtheria  toxin.  In  practice,  the  bacilli  are 
cultivated  in  bouillon.  The  cultures  are  freed  from  all  living 
bacilli  by  nitration.  The  liquid  nitrate  contains  the  toxin. 
This  nitrate  is  injected  into  healthy  susceptible  animals,  in 
increasing  doses.  Usually  the  horse  is  used,  since  large  quan- 
tities of  blood  can  be  drawn  from  this  animal  on  account  of  its 
size,  and,  moreover,  the  horse  is  very  susceptible.  Insuscep- 
tible animals  cannot  be  made  to  yield  antitoxin,  at  least  of 
any  appreciable  strength.  Eventually  enormous  doses  of 
toxin  are  given,  and  the  animal  acquires  a  high  degree  of  immu- 
nity. The  blood  of  the  animal  is  withdrawn,  taking  care  to 
avoid  contamination,  and  the  serum  allowed  to  separate  in  the 
refrigerator.  The  serum  of  the  blood  is  drawn  off  and  consti- 
tutes the  antitoxin.  The  use  of  antitoxin  has  been  eminently 
successful  and  revolutionized  the  treatment  of  diphtheria;  and 
it  has  given  complete  success  as  a  prophylactic  in  tetanus  with 
an  antitoxin  prepared  by  injecting  horses  with  increasing 
amounts  of  tetanus  toxin.  (See  the  description  of  the  bacteria 
of  these  diseases.) 

Ehrlich  discovered  that  the  vegetable  toxins,  abrin  and  ricin, 
behave  in  a  manner  very  similar  to  soluble  bacterial  poisons  when 
injected  into  animals,  and  that  by  their  injection  an  immunity 
for  the  same  poisons  may  be  secured.  Ehrlich  also  found  that 
the  milk  of  animals  which  had  been  immunized  with  increasing 
doses  of  abrin  and  ricin  confers  immunity  upon  sucklings. 
14 


210  MANUAL    OF    BACTERIOLOGY. 

There  is  little,  if  any,  analogy,  on  the  other  hand,  between 
the  tolerance  acquired  by  gradual  dosage  from  bacterial  and 
other  toxins  and  that  which  victims  of  the  morphine  and 
cocaine  habits  have  for  immense  doses  of  these  drugs,  for  no 
bodies  resembling  antitoxins  are  obtained  from  animals  that 
have  been  accustomed  to  such  drugs. 

The  gradual  injection  of  animals  with  bacteria  which  produce 
endotoxins,  B.  typhosus,  B.  choleras  Asiaticae,  B.  coli  communis 
and  others,  does  not  produce  antitoxin.  It  sometimes  seems 
to  have  no  effect,  at  others  it  seems  to  increase  the  bacteriolytic 
power  of  the  blood  for  the  bacteria  with  which  the  animal  is 
injected.  Frequently  it  has  a  most  peculiar  effect.  This 
peculiarity  was  first  shown  by  Loeffler  and  Abel*  in  experi- 
ments upon  guinea-pigs  injected  with  colon  bacilli  at  the  same 
time  given  injections  of  colon-immune  dog's  serum.  The  very 
unexpected  result  was  obtained  that  those  guinea-pigs  which 
received  smaller  doses  of  the  immune  serum  were  protected, 
whereas  those  injected  with  larger  doses  were  not  protected. 
Similarly,  Neisser  and  Wechsberg  found  in  test-tube  experi- 
ments with  cultures  that  the  serum  from  the  injected  animal 
sometimes  kills  the  kind  of  bacteria  used  for  injection  when 
it  is  diluted  but  not  when  it  is  undiluted.  This  action  is  so 
paradoxical  that  it  may  well  be  stated  in  other  words  for 
emphasis.  The  normal  blood-serum  of  uninoculated  animals, 
as  has  been  previously  stated,  is  bactericidal  for  many  bacteria. 
Now,  if  an  animal  furnishing  normally  highly  bactericidal 
serum  is  injected  with  an  endotoxic  bacterium,  its  blood-serum 
often  fails  to  kill  the  [kind  of  bacterium  with  which  it  is  in- 
jected, unless  the  serum  is  greatly  diluted.  It  still  retains  its 
power  to  kill  other  kinds  of  bacteria.t  The  peculiar  behavior 

*Loeffler,  F.,  and  Abel,  R.  Ueber  die  spezifischen  Eigenschaften  der  Schutz- 
korper  im  Blute  typhus-  und  coli-immuner  Tiere.  Cent.  f.  Bakt.,  Abt.  i,  Bd. 
19,  No.  2,  3,  p.  51-70.  Jena,  Jan.  23,  1896. 

fB.  H.  Buxton.  Bacteriolytic  Power  of  Immune  Serum  and  the  Theory  of 
Complement  Diversion.  Journal  of  Medical  Research,  V.  XIII.,  No.  5.  (Whole 
No.  90,  N.  S.  V.  S.)  p.  431-485.  Boston,  Aug.  1905. 


IMMUNITY.  211 

of  this  so-called  immune  serum  is  called  the  Neisser-Wechs- 
berg*  phenomenon.  The  theoretical  explanation  of  this  phe- 
nomenon will  be  discussed  below  under  Ehrlich's  side-chain 
theory. 

Buxtonf  reports  that  the  immunization  of  a  rabbit  with 
typhoid  cultures  does  not  enable  this  animal  to  dispose  of  a 
sublethal  dose  of  the  bacilli  any  more  quickly  than  the  normal 
rabbit  and  that  such  injections  do  not  produce  any  appreciable 
increase  of  resistance  to  the  endotoxin  of  the  typhoid  fever 
bacillus. 

Analogous  to  the  hypersensibility  to  infection  sometimes 
noted  on  the  injection  of  animals  with  repeated  doses  of  bac- 
teria is  the  phenomenon  of  anaphylaxis  seen  in  animals  injected 
with  repeated  dose  of  foreign  serum.  Animals  injected  with 
egg  albumin  or  with  blood-serum  derived  from  some  other 
species  withstand  an  amount  of  the  albumin  at  the  first  injection 
which  kills  them  on  subsequent  injection.  Rosenau  and 
Anderson  J  have  found  that  the  reaction  of  the  animals  in  this 
case  is  specific.  That  is,  guinea-pigs  sensitized  with  horse 
serum  are  very  sensitive  to  a  second  injection  with  horse  serum, 
but  very  slightly  susceptible  to  injection  with  the  serum  from 
other  animals  such  as  rabbit,  cat,  dog,  hog,  sheep,  chicken  or 
man.  Furthermore,  a  guinea-pig  may  be  rendered  sensitive 
to  three  different  kinds  of  proteids  at  once  by  injecting  the  pro- 
teids  either  all  simultaneously  or  all  separately. 

Active  and  Passive  Immunity. — The  kind  of  immunity 
which  results  from  the  injection  of  substances  from  immu- 
nized animals  is  called  "  passive  immunity. "  Diphtheria  and 
tetanus  antitoxins  produce  passive  immunity.  "Active  im- 

*Max  Neisser  and  Friedrich  Wechsberg.  Ueber  die  Wirkungsart  Bacteri- 
cider  Sera.  Milnchner  Med.  Woch.  Jahrg.  48,  No.  13,  p.  697-700.  Munch. 
Apr.  30,  1901. 

t  Journ.  Med  Research,  V.  XVI.,  No.  2,  May,  1907.     pp.  251-274. 

tHyg.  Lab.  Bui.  No.  29  and  Bui.  No.  36.  U.  S.  Public  Health  and  Marine 
Hosp.  Serv.  April,  1906  and  1907  resp.  Also  Journ.  Infec.  Dis.  Vol.  IV., 
1907.  pp.  552-557. 


212  MANUAL    OF    BACTERIOLOGY. 

munity, "  on  the  other  hand,  is  that  which  results  in  some 
cases: 

(i)  From  a  spontaneous  attack  of  an  infectious  disease; 
(2)  from  an  attack  excited  artificially  through  inoculation  with 
small  doses  of  virulent  cultures;  (3)  from  the  administra- 
tion of  large  doses  of  attenuated  cultures;  (4)  from  the 
injection  of  bacterial  products  (toxins)  freed  from  the  bacteria 
themselves.  Pasteur's  methods  of  protective  inoculation  for 
anthrax  and  other  diseases,  and  Haffkine's  injections  for  bu- 
bonic plague,  the  gradual  injection  of  cultures  of  diphtheria  or 
of  tetanus  bacilli  into  animals,  produce  active  immunity. 
Active  immunity  is  usually  more  enduring  than  passive  immu- 
nity. But  passive  immunity,  resulting,  as  it  does,  from  the 
direct  introduction  of  antitoxin,  is  brought  about  more  quickly 
than  active  immunity. 

THEORIES    OF    IMMUNITY. 

Phagocytosis.* — Metchnikoff  described  under  the  name 
"phagocytosis"  immunity  and  recovery  from  bacterial  inva- 
sion. This  theory  is  based  on  the  well-known  fact  that  cer- 
tain cells  of  the  body  have  the  power  of  surrounding  and  in- 
gesting foreign  substances.  The  cells  in  question  are  chiefly 
polynuclear  leukocytes,  but  to  some  extent  other  leukocytes 
and  endothelial  and  other  cells  are  also  concerned.  The  poly- 
nuclear  leukocytes  are  the  cells  which  destroy  bacteria,  and 
Metchnikoff  now  calls  these  microphages;  other  phagocytes  he 
calls  macrophages.  There  are  many  examples  of  phagocytosis 
which  have  been  observed.  The  phagocytes  of  the  lungs  con- 
stantly take  up  small  bits  of  carbon  inhaled  with  the  air.  Par- 
ticles of  carmine  injected  into  the  tissues  will  later  be  found 
within  phagocytes.  After  a  hemorrhage,  phagocytic  cells  may 
be  found  containing  red  blood-corpuscles  or  particles  of  blood- 

*Greek,  ^cryetV,  to  eat;  KI/TOS,  a  cell. 


IMMUNITY.  213 

pigment.  The  presumption  is  that  phagocytic  cells  serve  to 
remove  irritating  and  foreign  bodies  and  to  destroy  them. 
Metchnikoff  showed  that  phagocytes  also  absorb  bits  of  degen- 
erating or  useless  tissue.  Such  particles  disintegrate,  and  they 
are  digested  and  become  a  part  of  the  protoplasm  of  the 
phagocytes.  This  process  is  seen  when  the  tail  of  the  tadpole 
shortens.  The  superfluous  part  is  absorbed,  at  least  in  part, 
by  phagocytic  leukocytes.  Metchnikoff' s  earlier  observations 
were  made  largely  on  the  invertebrates,  whose  transparent 
bodies  may  be  studied  while  living.  One  illustration  was 
furnished  by  a  small  crustacean  (Daphnia  or  water-flea,) 
which  was  often  infected  with  a  fungus.  Some  infected  in- 
dividuals died,  others  recovered.  Metchnikoff  found  that 
the  cells  of  the  fungus  might  be  ingested  and  destroyed  by  the 
leukocytes  of  the  Daphnia.  He  described  the  history  of  this 
disease  as  a  contest  between  the  parasitic  cells  and  the  phago- 
cytes, in  which  either  might  succeed.  Similarly,  when  anthrax 
bacilli  were  introduced  into  frogs,  which  are  immune  from 
anthrax,  the  bacilli  were  ingested  by  the  frog's  leukocytes. 
Metchnikoff*  contends  that  this  function  of  leukocytes  and 
other  phagocytic  cells  constitutes  the  principal  defense  of  the 
body  against  bacteria. 

Other  investigators  also  have  seen  bacteria  inclosed  within 
the  bodies  of  leukocytes.  It  has  been  urged  by  some  that  the 
bacteria  are  already  dead  when  the  leukocytes  devour  them, 
but  Metchnikoff  showed  that  these  inclosed  bacteria  are  still 
alive,  for  they  produce  disease  when  introduced  into  fresh  ani- 
mals; so  they  are  apparently  not  injured  before  they  are  taken 
up.  In  other  cases,  as  with  the  gonococcus,  which  is  com- 
monly found  inclosed  within  leukocytes,  it  is  quite  evident 
from  their  appearance  that  the  bacteria  retain  their  full  vigor 
after  being  ingested. 

*Metchnikoff.  Comparative  Pathology  of  Inflammation.  Trans.,  Starling. 
1893. 


214  MANUAL    OF    BACTERIOLOGY. 

It  is  well  known  that  a  suppurating  part  contains  large 
numbers  of  leukocytes,  and  one  of  the  most  characteristic 
events  in  the  inflammatory  process  is  the  migration  of  leuko- 
cytes to  the  point  of  irritation.  This  indicates  a  positive 
chemotaxis  for  leukocytes  on  the  part  of  substances  in  the  in- 
flamed area.  Metchnikoff  believes  that  the  function  of 
these  leukocytes  is  to  destroy  the  bacteria  and  to  arrest  their 
further  progress.  On  this  theory  bacteria  have  often  been 
likened  to  an  invading  army  and  the  leukocytes  or  phagocytes 
to  a  force  designed  to  repel  their  attacks. 

It  is  certain  that  in  some  infectious  diseases  the  number  of 
leukocytes,  chiefly  of  the  polynuclear  neutrophilic  variety,  in 
the  circulating  blood  is  increased  (leukocytosis).  This  is  the 
case  usually  in  lobar  pneumonia  and  acute  suppurative  in- 
fections. In  some  other  infectious  diseases  there  is  usually 
no  leukocytosis;  for  example,  tuberculosis,  typhoid  fever  and 
malaria.  It  is  interesting  to  observe  that  in  trichinosis,  and 
more  rarely  in  infection  with  other  animal  parasites,  the 
eosinophilic  leukocytes*  become  much  more  numerous  in  the 
blood  than  normally. 

Manwaring  and  Ruh|  studied  the  effects  of  various  anti- 
septic and  therapeutic  agents  upon  phagocytosis,  and  found: 
That  carbolic  acid  causes  diminution  of  phagocytosis  in  pro- 
portion to  the  amount  of  carbolic  acid  present,  causing  complete 

*  Williams  and  Bentz.  Trans.  Association  of  American  Physicians.  XVIII. 
1903.  p.  152. 

FURTHER  REFERENCES. 

Hektoen.     Jour.  Amer.  Med.  Assn.     May  12,  1906. 
Simon.     Jour.  Exper.  Med.  VIII.,  No.  6  and  IX.,  No  5. 
Various  articles.     Jour.  Exper.  Med.     Vol.  IX. 
Potter,  etc.     Jour.  Amer.  Med.  Assn.     Nov.  30,  1907. 
Wright.     Jour.  Amer.  Med.  Assn.     Aug.  10  and  17,  1907. 
Ross,  etc.     Jour.   Amer.   Med.   Assn.     Oct.    12,    1907. 
Wright,  etc.     Lancet.     Nov.  2,  1907. 
Park  and  Biggs.     Jour.  Med.  Res.     Vol.  XVII.,  No.  i. 
Exper.  Med.     Vol.  IX.     pp.  473-486.      1907 


IMMUNITY.  215 

cessation  when  present  in  the  proportion  of  J  per  cent.  That 
corrosive  sublimate  in  the  proportion  of  less  than  T^Q-  per 
cent,  causes  preliminary  stimulation  followed  by  gradual 
diminution.  In  larger  amounts  it  causes  deterioration  from 
the  start.  Boric  acid  less  than  ij  per  cent,  causes  transient 
stimulation,  followed  by  depression  and  complete  cessation  at 
2  per  cent.  Quinine  hydrochloride  causes  stimulation  of 
phagocytosis  up  to  y^-  per  cent.,  which  is  the  maximum  effect. 
Larger  amounts  are  depressing.  Complete  cessation  at  TV 
per  cent. 

Experiments  have  been  made  by  various  investigators, 
consisting  in  the  production  of  local  leukocytosis,  and 
studying  the  effects  of  the  leukocytes  thus  brought  to- 
gether upon  bacteria.  The  injection  into  the  pleural  or  .peri- 
toneal cavity  of  various  substances,  notably  nucleic  acid,  or 
aleuronat  suspensions,  calls  forth  a  great  accumulation  of 
leukocytes,  and  these  masses  of  leukocytes  have  been  used  for 
the  purpose  of  observing  the  phenomena  of  phagocytosis  both 
inside  and  outside  the  body.* 

In  operations  upon  the  abdominal  cavity  the  production  of 
artificial  leukocytosis  in  the  peritoneal  cavity  previous  to 
operations  has  been  suggested  and  tried  with  apparent  success 
on  the  ground  that  if  any  bacteria  entered  during  the  operation 
they  would  be  destroyed  by  the  phagocytes. f 

Rubin  I  found  that  narcotics,  alcohol,  ether,  chloroform,  ap- 
pear to  directly  lessen  the  affect  of  the  substances  which  in- 
hibit the  growth  and  toxic  action  of  the  bacteria  in  the  bodies 
of  normal  animals. 


*Gustav  F.  Ruediger.  The  Mechanism  of  Streptococcus  Infection.  Journal 
of  the  American  Medical  Association.  No.  3,  January  21,  1905.  p.  198. 
L  idvig  Hektoen  and  Gustav  F.  Ruediger.  Studies  in  Phagocytosis.  Journal 
of  Infectious  Diseases.  Vol.  II.,  No.  i,  p.  128  January,  1905. 

fVon  Mikulicz.  Versuche  iiber  Resistenzvermehrung  bei  Magen  und  Darm- 
perforationen.  Archiv  fur  klinische  Chirurgie.  Bd.  LXXIIL,  Heft  2,  p.  347. 
1904. 

iJourn.     Infectious  Diseases.     Vol.  I.,  1904.     pp.  425-444. 


2l6  MANUAL    OF    BACTERIOLOGY. 

On  the  other  hand,  Wright  and  Douglas,*  whose  results  have 
been  corroborated  by  others,  found  that  certain  substances  pre- 
pare the  bacteria  for  the  phagocytes.  These  substances  are 
developed  in  the  blood  under  certain  conditions.  Thus,  in- 
jections of  dead  cultures  of  the  S.  pyogenes  aureus  into  the 
blood  produces  a  substance  which  prepares  the  live  staphy- 
lococcus  as  food  for  the  phagocytes.  Substances  acting  in  this 
way  they  call  "opsonins"  (opsono,  I  prepare  victuals  for). 

These  authors  find  that  phagocytosis  for  certain  organisms 
depends  upon  the  presence  of  opsonins  in  the  blood.  Thus, 
B.  typhosus,  S.  choleras  Asiaticae,  B.  coli  communis,  B.  dysen- 
teriae,  S.  pyogenes  aureus,  B.  pestis,  M.  melitensis,  D.  pneu- 
monias, are  all  taken  up  by  phagocytes  after  being  prepared  by 
the  opsonins.  B.  diphtherias  and  B.  xerosis  are  not  acted  upon 
by  opsonins. 

Wright  speaks  of  the  number  of  bacteria  which  may  be 
counted  inside  of  the  leukocytes  on  an  average  as  the  "opsonic 
index,"  and  he  uses  so-called  vaccines  derived  from  cultures 
with  the  purpose  of  increasing  this  index.  There  is  wide 
diversity  of  opinion  in  regard  to  the  value  of  the  whole  opsonic 
theory,  both  for  diagnostic  and  for  therapeutic  purposes,  and 
the  stronger  authority  seems  opposed  to  attributing  very  great 
value  to  the  use  of  opsonins  for  therapeutic  purposes. 

Hektoent  finds  that  the  opsonic  function  of  normal  and 
immune  serum  is  due  to  a  distinct  body,  different  from  lysins 
and  agglutinins. 

BuxtonJ  finds  support  for  Wright's  view  that  the  opsonins 
are  a  very  important  factor  in  immunity.  The  influence  of  the 
opsonins  on  the  bacteria  is  to  make  them  more  acceptable  to 


*A.  E.  Wright  and  Stewart  R.  Douglas.  An  Experimental  Investigation  of 
the  Role  of  the  Blood  Fluids  in  Connection  with  Phagocytosis.  Proceedings 
of  the  Royal  Society.  Vol.  LXXIL,  No.  483,  p.  357.  October  31,  1903. 
Ibid.  Vol.  LXXIIL,  No.  490,  p.  128.  February  and  March,  1904. 

fHektoen.     Journ.  Infectious  Diseases.     Vol.  Ill,  1906.     pp.  434-440. 

%Journ.  Med.  Research.     V.  XVI.,     No.   2,   1907.     p.   264. 


IMMUNITY.  217 

the  makrophages ;  with  this  influence,  however,  the  power  of  the 
opsonins  seems  to  terminate,  for  the  makrophages  of  the  im- 
mune animal  do  not  seem  to  destroy  the  bacilli  more  quickly 
than  the  normal  one.  It  may  be  that  the  opsonins  indirectly 
protect  the  immune  animal,  since  where  phagocytosis  is  active 
the  endotoxins  are  liberated  inside  the  phagocytes,  and  not  in 
the  circulating  plasma. 

Neufeld  and  Rimpau*  found  that  antistreptococcus  serum 
acts  as  an  opsonin. 

Just  the  contrary  effect  to  opsonins,  on  the  other  hand,  is 
produced  by  certain  other  bodies;  for  Bailf  has  found  that 
when  tubercle  bacilli  undergo  bacteriolysis  certain  substances 
are  liberated  which  check  phagocytosis.  These  he  regards  as 
endotoxins,  and  gives  to  them  the  name  aggressins. 

There  can  be  no  doubt  but  that  phagocytosis  plays  an  im- 
portant part  in  combating  bacterial  infection.  And  what  fol- 
lows in  regard  to  the  germicidal  and  antitoxic  action  of  the 
fluid  portion  of  the  blood  containing  no  phagocytes  does  not 
alter  the  fact  that  phagocytes,  when  these  are  present,  do  de- 
"stroy  the  bacteria.  Moreover,  Metchnikoff  maintains  that 
the  germicidal  property  of  the  blood-serum  free  from  cells  is 
due  to  substances  liberated  from  the  phagocytes  by  phagolysis, 
or  breaking  up  of  phagocytes.  In  other  words,  he  holds  that 
infection  is  combated  by  the  phagocytes  or  by  substances  de- 
rived from  the  phagocytes. 

But  be  this  as  it  may,  it  is  well  established  that  the  serum  of 
the  blood  deprived  of  leukocytes  also  has  the  property  of  de- 
stroying bacteria  in  many  cases.  The  effect  produced  upon 
the  bacteriolytic  property  of  the  blood-serum  by  injecting  the 
animal  from  which  the  serum  is  obtained  with  bacterial  cul- 
tures has  already  been  stated  (page  210). 

*Deutsche  medicinische  Wochenschrijt.     1904.     Bd.  XXX.,  p.    1458. 
fOskar   Bail.     Wiener   klinische    Wochenschrift.     Bd.    XVIII.,    1905.     Re- 
view in  Bulletin  de  Vlnstitut  Pasteur.     III.,     p.  348.     1905. 


2l8  MANUAL    OF    BACTERIOLOGY. 

It  has  already  been  explained  that  the  neutralization  of 
bacterial  poisons  or  toxins  takes  place  by  the  production  of 
antitoxins  (page  209),  and  that  the  gradual  injection  of  toxins 
is  followed  by  a  greatly  increased  production  of  antitoxin. 
Bacteriolysins,  then,  may  be  produced — or  at  least  their  pro- 
duction may  be  stimulated — by  injections  of  bacteria,  while 
antitoxins  are  produced  by  injections  of  toxins;  and  Ehrlich 
advanced  his  now  celebrated  side-chain  theory  to  explain  these 
phenomena  as  well  as  the  formation  of  agglutinins,  lysins  and 
precipitins. 

Ehrlich's  Side-chain  Theory  of  Immunity.*— Ehrlich 
was  the  first  to  offer  an  explanation  from  a  chemical  point  of 
view  of  the  action  of  toxins  on  cellular  protoplasm  and  the  for- 
mation of  antitoxins.  He  assumes,  to  begin  with,  that  the 
molecules  of  the  protoplasm  are  to  be  regarded  as  being  en- 
dowed with  chemical  groups,  present  in  the  form  of  lateral  ap- 
pendages to  the  molecule,  called  side-chains.  They  can  be 
illustrated  by  the  analogies  presented  by  the  graphically  writ- 
ten formulae  of  some  complex  molecules.  It  is  necessary  to 
conceive  of  molecules  made  of  an  immense  number  of  atoms, 
and  bristling  with  projecting  side-chains.  The  function  of  the 
side-chains  is  to  become  attached  to  other  organic  molecules 
with  which  they  have  affinities.  In  this  manner  they  aid  in 
absorbing  the  substances  essential  for  the  nutrition  of  the 
protoplasm  of  cells. 

The  side-chains  are  therefore  also  called  "receptors" — a  more 
appropriate  name.  The  numerous  receptors  which  a  molecule 

*The  literature  of  this  subject  is  very  extensive.  An  exhaustive  review  is 
that  by  L.  Aschoff.  Ehrlich's  Seitenkettentheorie.  Zeitschrift  fur  allgemeine 
Physiologic .  1902. 

The  following  are  also  of  a  general  character:  Ehrlich's  Croonian  Lecture. 
Proceedings  of  the  Royal  Society.  LXIL,  p.  437.  1900.  Ehrlich.  Schluss- 
betractungen.  Nothnagel's  System  of  Medicine.  Vol.  VIII.  H.  C.  Ernst. 
Modern  Theories  of  Bacterial  Immunity.  1903.  Prudden.  Medical  Record. 
February  14,  1903.  Ritchie.  Journal  of  Hygiene.  Vol.  II.  1902.  Bergey. 
American  Medicine.  October  n,  1902.  Immunity.  Special  Article.  Journal, 
o}  the  American  Medical  Association.  No.  4  et  seq.  1901;. 


IMMUNITY. 


219 


has  are  of  many  kinds,  with  affinities  for  other  molecules  of 
different  kinds.  Each  kind  of  receptor  will  then  have  an 
affinity  for  a  molecule  of  a  particular  kind,  which  it  may  be 
said  to  "fit,"  as  a  key  fits  in  a  lock,  although  this  expression 
must  not  be  taken  in  a  literal  sense.  A  receptor  to  which 
tetanus  toxin  might  become  attached  would  not  "fit"  diphtheria 
toxin.  In  order  that 
toxins  may  be  able  to 
combine  with  the  recep- 
tors, their  structure  must 
be  nearly  like  that  of  the 
food  molecules  which 
the  receptors  are  adapted 
to  receive. 

Secondly,  soluble 
toxins  are  to  be  looked 
upon  as  definite  chemical 
bodies  excreted  by  bac- 
teria, and  containing  two 
essential  groups  of 

atoms.  One  group  is 
the  haptophore,  by  means 
Of  Which  the  toxin  may 
be  linked  with  the  re- 


FIG.    50.—  Receptors     of    the    first   order 
j^,  ™th  ^.-(Journal  of  the  American 
Medical  Association.   1905.  P.  955.) 

a.  Cell  receptor,      b.  Toxin  molecule,     c. 
Haptophore  of  the  toxin  molecule,     d.  Toxo- 
phore  of  the  toxin  molecule,     e.  Haptophore 
ceptors  of  the  molecules       of  the  cell  receptor. 

of  the  cell.     The  other 

group  is  the  toxophore,  which  is  capable  of  destroying  the 
protoplasmic  molecule,  after  attached  to  the  receptor  of  the 
latter  by  the  haptophore. 

These  relations  have  been  represented  schematically.  In 
Fig.  50  a  portion  of  a  cell  is  shown,  with  receptors.  A  m  )lecule 
of  toxin,  b,  is  attached  by  its  haptophore,  c,  to  the  haptophore 
of  the  cell  receptor,  a.  A  free  cell  receptor  is  also  shown  with 
its  haptophore,  e,  capable  of  uniting  with  any  toxin  molecule 


220  MANUAL    OF    BACTERIOLOGY. 

that  may  be  present.  The  toxin  molecule,  b,  has  its  toxophore 
group  represented  by  the  fringe-like  end,  d.  If  the  cell  receptor 
becomes  detached  from  the  cell,  its  haptophore,  e,  may  unite 
with  a  toxin  molecule  as  readily  as  when  the  receptor  is  still 
attached  to  the  cell.  Such  a  detached  receptor  constitutes  a 
molecule  of  antitoxin. 

As  the  side-chains  or  receptors  of  the  protoplasm  are  es- 
sential to  its  existence,  their  combination  with  the  toxin, 
through  its  haptophore,  results  in  destruction  of  the  molecule. 
But  if  the  damage  be  not  too  serious,  the  protoplasm  is  stimu- 
lated to  produce  numerous  similar  side-chain  groups — to  an 
overproduction  of  these,  in  fact.  As  not  all  of  these  are  neces- 
sary for  the  performance  of  its  functions,  the  superfluous  ones 
are  thrown  off  into  the  surrounding  serum.  It  is  well  known 
that  many  cells  of  the  body  exhibit  analogous  heightened  acti- 
vities under  stimulating  influences,  as  pointed  out  by  Weigert. 
If  such  free  side-chains  or  receptors  combine  with  the  hap- 
tophorous  groups  of  the  toxin,  the  latter  is  no  longer  able  to 
combine  with  the  protoplasm  of  the  cells.  Thus  they  act  as  a 
kind  of  buffer  in  protecting  the  protoplasm  from  the  attacks  of 
the  toxins.  These  free,  cast-off  receptors  constitute  the 
antitoxic  part  of  the  serum  as  stated. 

Numerous  experiments  have  been  made  which  illustrate 
the  probable  chemical  nature  of  antitoxin  action.  A  fatal 
dose  of  diphtheria  or  tetanus  toxin  may  be  neutralized  outside 
of  the  body  by  mixing  it  with  its  appropriate  antitoxin.  In- 
jection of  the  mixture  shows  it  to  be  innocuous  to  animals. 

The  manner  in  which  toxins  combine  with  protoplasm  has 
been  shown  in  the  case  of  tetanus  toxin.  The  nitrate  from 
cultures  of  tetanus  bacilli  will  kill  guinea-pigs,  presumably 
by  damage  to  the  central  nervous  system.  The  same  filtrate 
rubbed  up  with  brain  or  spinal  c  ^rd  has  been  found  to  have  lost 
its  toxic  properties.  It  may  be  assumed  that  the  poison  has 
combined  with  the  protoplasm  of  the  cells. 


IMMUNITY. 


221 


r-f 


But  the  side-chain  theory  offers  an  explanation  not  only  of 
the  mechanism  of  the  union  of  toxin  and  antitoxin,  but  also  ex- 
plains the  phenomena  of  agglutination,  precipitation  and 
cytolysis.  In  the  union  of  antitoxin  and  toxin,  as  stated  above, 
the  process  is  a  simple  combining  of  the  toxin  with  the  recep- 
tor, and  there  the  process  ends.  Receptors  of  this  kind  are 
called  receptors  of 
the  first  order  (Fig. 
50).  But  after  the 
union  of  the  agglu- 
tinins  and  of  the  pre- 
cipitins  with  their  re- 
ceptors further  change 
takes  place.  In  the 
one  case,  clumping; 
in  the  other,  precipi- 
tation;  and  these 
changes  are  brought 
about  by  a  kind  of 
fermentative  action. 
So,  in  addition  to  the 
haptophore  group, 
the  receptor  must 
possess  a  ferment- 
producing  group.  It 
seizes  on  the  red  cells 


FIG.  51. — Receptors  of  the  second  order  and 
of  some  substance  uniting  with  one  of  them. — 
(Journal  of  the  American  Medical  Association. 
1905.  P.  1113.) 

c.  Cell  receptor  of  the  second  order,  d.  Tox- 
ophore  or  zymophore  group  of  the  receptor. 
e.  Haptophore  of  the  receptor.  /.  Food  sub- 
stance or  product  of  bacterial  disintegration 
uniting  with  the  haptophore  of  the  cell  receptor. 


or  on  the  bacteria,  as 

the  case  may  be,  with  the  haptophore  group,  and  produces 
•certain  changes  with  its  ferment-producing  group.  The 
latter  is  called  the  zymophore  group.  Receptors  of  this  kind 
are  called  receptors  of  the  second  order  (Fig.  51). 

With  the  lysins  there  is  also  a  change,  which  takes  place  after 
the  receptor  unites  with  the  bacteria  or  other  cells;  so  there 
must  be  here  also  a  zymophore  or  zymotoxic  group,  as  it  is 


222 


MANUAL    OF    BACTERIOLOGY. 


called.  This  zymotoxic  group,  however,  is  not  an  integral 
part  of  the  receptor,  but  is  easily  broken  off  from  it,  in  the 
manner  described  below. 

As  is  explained  later,  the  power  of  the  lysins  becomes  sus- 
pended, but  not  lost,  on  being  heated  to  55°  or  56°  C.  In  this 
condition  they  are  said  to  be  inactivated.  They  become  active 
again  when  certain  fresh  serum  is  added — not  necessarily  fresh 


FIG   52. — Receptor  of  the  third  order,  and  of  some  substance  uniting  with  one 
of  them. — (Journal  of  the  American  Medical  Association.     1905.  P.  1369.) 
c.  Cell  receptor  of  the  third  order — an  amboceptor.     e.  One  of  the  hapto- 
phores  of  the  amboceptor,  with  which  some  food  substance  or  product  of  bac- 
terial disintegration  (/)  may  unite,     g.  The  other  haptophore  of  the  ambo- 
ceptor with  which  complement  may  unite,     k.  Complement,     h.  The  hapto- 
phore.    z.  The  zymotoxic  group  of  complements. 

lysin,  but  fresh  normal  serum.  This  will  all  be  discussed 
and  explained  later.  For  the  present  purpose  it  is  sufficient 
to  bear  in  mind  that  lysin  becomes  inactivated  and  may  be 
reactivated.  So  the  lysins  act  differently  from  agglutinins  and 
precipitins.  They  must  have  peculiar  receptors  which  unite, 
on  the  other  hand,  with  the  cells  which  they  disintegrate  and, 
on  the  other,  with  a  ferment-producing  substance  easily  de- 
stroyed by  heat.  These  receptors  must  possess  two  haptophore 


IMMUNITY.  223 

groups;  in  other  words,  a  haptophore  for  cells  and  a  hapto- 
phore  capable  of  uniting  with  a  body  containing  a  ferment- 
producing  group.  Receptors  for  lysins  are  therefore  called 
amboceptors,  or  receptors  with  two  haptophores  (Fig.  52). 
They  are  also  called  receptors  of  the  third  order.  The  sub- 
stance which  reactivates  the  lysin,  the  fresh  serum,  is  called 
complement,  and  it  must  be  composed  of  a  haptophore  in 
order  to  attach  itself  to  the  amboceptor,  and  a  zymotoxic 
group  in  order  to  produce  lysis.  On  heating  fresh  normal 
serum  to  55°  or  56°  C.  the  complement  which  it  contains  is  not 
destroyed,  but  its  zymotoxic  group  alone  is  destroyed;  the 
haptophore  group,  on  the  other  hand,  resists  heat.  So  if 
heated  complement  be  added  to  inactivated  lysin,  it  unites 
with  the  freed  haptophore.  A  lysin  inactivated  by  heat  with 
fresh  serum  added  disintegrates  homologous  cells;  but  a  lysin 
inactivated  by  heat  when  heated  fresh  serum  is  added  will  not 
only  not  produce  lysis  of  homologous  cells,  but  will  not  do  so 
even  when  unheated  fresh  serum  is  subsequently  added. 

The  behavior  of  mixtures  of  toxins  and  antitoxins  is  most  peculiar,  for  they 
do  not  in  all  cases  obey  the  simple  rule  of  relative  proportion.  It  is  true  that 
if  a  certain  amount  of  antitoxin  neutralizes  a  certain  amount  of  toxin,  then  any 
multiple  of  this  amount  of  antitoxin  will  neutralize  the  same  multiple  of  toxin  if 
the  two  are  mixed  all  at  once.  So  far  the  rule  is  simple.  But  if  100  doses  of 
toxin — i.  e.,  enough  to  kill  100  guinea-pigs—is  exactly  neutralized,  and  then  the 
amount  of  free  toxin  necessary  to  kill  a  guinea-pig  is  added,  it  will  not  kill  a 
guinea-pig  as  would  be  expected.  Many  doses  have  to  be  added,  sometimes 
as  much  as  thirty  or  forty  doses  or  more,  before  the  mixture  again  becomes 
lethal. 

Another  remarkable  property  is  that  toxin  that  has  stood  for  a  long  time 
loses  greatly  in  poisonous  properties,  but  not  in  its  power  of  combining  with 
antitoxin.  Furthermore,  this  old  toxin  will  produce  antitoxin  if  injected  into 
horses  or  other  susceptible  animals. 

In  order  to  explain  these  extraordinary  reactions  several  theories  have  been 
advanced,  and  in  this  connection  certain  peculiar  reactions  obtained  with 
lysins,  agglutinins  and  precipitins,  which  have  helped  to  give  an  insight  into 
the  processes  involved,  have  also  been  explained  on  similar  theoretical  grounds. 
These  theories  will  now  be  discussed. 

Ehrlich  regards  the  beef-broth  from  a  diphtheria  or  tetanus  culture  as  a 


224 


MANUAL    OF    BACTERIOLOGY. 


solution  of  several  different  but  related  bodies,  and  he  makes  so-called  "spectra" 
to  explain  this  idea.  He  thinks  that  primarily  the  substances  are  toxin  and 
toxon  (Fig.  53),  each  having  affinity  for  antitoxin;  but  the  affinity  of  toxon  for 
antitoxin  is  weaker  than  the  affinity  of  toxin  for  the  antitoxin.  And,  further- 
more, toxon — no  matter  in  what  dose — does  not  kill  guinea-pigs  quickly  if  at 
all,  but  causes  a  paralysis  of  the  animal  some  weeks  after  inoculation,  while 
toxin,  on  the  other  hand,  in  just  the  proper  amount  kills  a  guinea-pig  weighing 
250  grams  in  two  days.  This  is  the  standard  minimum  fatal  dose,  or  i  d.  1. 
(dosis  letalis). 

Now,  if  enough  antitoxin  is  added  to  the  poisonous  beef-broth  it  will  neutral- 
ize both  the  toxin  and  the  toxon,  but  if  not  enough  is  added  for  this,  the  toxin 
is  first  neutralized  and  the  toxon  still  produces  paralysis  of  the  guinea-pig. 

But  on  standing,  both  toxin  and  toxon  quickly  become  changed;  a  part  of  the 
toxin  is  converted  into  a  body  called  toxoid  and  a  part  of  the  toxon  into  toxonoid, 
and,  while  retaining  their  affinity  for  antitoxin,  these  are  both  weakened  in  patho- 
genic power  as  compared  with  the  original  toxin;  toxoid,  in  fact,  is  devoid  of 
toxic  properties.  Toxoid,  then,  is  toxin  deprived  of  its  toxophore,  but  it  retains 


Tox.on. 


200 


FIG.  53. — "Spectrum"  of  theoretically  fresh,  crude  toxin. 
Such  a  combination  probably  does  not  occur. 


the  haptophore  group.  Still  further,  the  resulting  toxoid  and  the  toxon  have 
each  three  different  degrees  of  affinity  for  antitoxin. 

A  part  of  the  toxoid  has  less  affinity  than  the  toxin;  a  part  equal;  a  part  more. 
These  are  designated  epitoxoids,  syntoxoids  and  protoxoids,  respectively  (Figs. 
54  and  55),  and  toxons  are  in  like  manner  designated  epitoxons,  syntoxons  and 
protoxons.  But,  since  all  toxons  have  less  affinity  for  antitoxin  than  toxin  has, 
it  follows  that  epitoxoid  and  toxon  are  the  same.  All  crude  toxin,  then,  is 
composed  of  a  mixture  of  toxin,  toxoid  and  toxon,  for  toxoids  begin  to  form 
immediately,  so  that  toxin-toxon  without  toxoid  is  not  known. 

When  enough  antitoxin  is  added  to  100  doses  (too  d.  l.'s)  of  crude  toxin 
to  just  neutralize  it,  all  the  toxin  and  all  the  toxon  are  united  to  antitoxin.  But 
if  fresh  toxin  is  added,  some  of  the  toxoid  and  toxon  is  liberated,  and  the  added 
toxin  becomes  attached  to  the  antitoxin  in  its  place ;  and  so  with  each  additional 
amount  of  toxin  added  more  toxon  and  toxoid  is  liberated  till  the  point  is  reached 
where  all  the  toxon  and  toxoid  is  free,  and  the  additional  toxin  finds  all  the 


IMMUNITY. 


225 


haptophores  of  antitoxin  occupied  by  the  toxin  previously  added.  In  this  case 
any  additional  toxin  remains  uncombined,  and,  if  such  a  mixture  is  injected 
into  a  guinea-pig,  the  animal  is  killed. 

Bordet's*  explanation  differs  from  Ehrlich's.  Bordet  does  not  admit  the  exist- 
ence of  toxons,  and  regards  the  paralysis  attributed  by  Ehrlich  to  the  action  of  this 
hypothetical  substance  as  due  to  weakened  toxin.  He  explains  the  peculiar  behav- 
ior of  a  neutralized  mixture  of  crude  toxin  with  antitoxin,  stated  above,  by  assuming 
that  antitoxin  is  capable  of  taking  up  and  neutralizing  varying  amounts  of  toxin. 


Pro I 'OK  Old 


Toxen. 


C  60  'CO  2.00 

FIG.  54. — "  Spectrum  "  of  very  fresh  crude  toxin. 

He  compares  the  effect  of  mixing  toxin  and  antitoxin  to  that  of  mixing  starch  and 
iodine:  the  more  iodine  added  to  the  starch,  the  bluer  the  color.  Let  A  repre- 
sent, then,  a  certain  amount  of  antitoxin;  let  A  be  capable  of  combining  r,  2,  3, 
4,  5,  different  amounts  of  toxin;  call  these  amounts  of  toxin  TI}  T2,  T3,  T4,  Ts; 
and  assume  that  a  combination  in  which  all  the  A's  are  combined  with  T's  in 


Synloxoid 


y////////////// 


0  6O  /GO  2&> 

FIG.  55. — "  Spectrum"  of  crude  toxin  as  it  is  supposed  practically 
always  to  occur. 

the  proportion  of  ATly  is  neutral,  that  it  has  no  poisonous  properties;  that  a 
combination  represented  by  AT2  also  has  no  toxic  properties,  but  that  AT3, 
would  begin  to  show  toxic  properties,  and  that  A  T4  is  distinctly  toxic,  and  that 
ATS  is  very  toxic.  Starting  with  toxin,  then,  if. just  enough  antitoxin  is  added 
to  neutralize  its  poisonous  properties,  AT-i  is  first  formed,  which  is  not  toxic; 
now  add  more  toxin,  and  none  of  this  remains  free,  but,  on  the  contrary,  A  T2  is 
formed,  which  is  not  toxic;  on  adding  still  more,  when  AT4  or  ATS  is  reached 

*Bordet.     Toxines    et    Antitoxines.     Annales   de   I'Institut   Pasteur.     1903. 
p.  161  et  seq. 

15 


226  MANUAL    OF    BACTERIOLOGY. 

the  mixture  is  fatal  for  guinea-pigs.  The  paralysis  which  Ehrlich  attributes  to 
toxon  would  be  represented,  say,  by  AT3.  In  between  the  the  combinations 
represented  by  AT-s.  to  ATS  are  all  imaginable  combinations,  a  sliding  scale 
of  no  definite  units. 

In  other  words,  while  Ehrlich  holds  that  toxin  and  antitoxin  unite  in  one 
definite  proportion.  Bordet  holds  that  they  may  unite  in  any  proportions,  like 
two  different  colors  of  paint  mixed  together  producing  any  intermediate  color 
with  more  or  less  tint  of  one  or  other  of  the  original  colors. 

The  evidence  adduced  by  Bordet  for  this  conception  is  very  abundant  and 
fully  repays  study. 

Still  another  theory  offered  to  explain  the  peculiar  behavior  of  the  antitoxin- 
toxin  mixture  is  advanced  by  Arrhenius  and  Madsen,  also  supported  by  experi- 
mental evidence.  They  also  deny  the  existence  of  toxon,  and  look  upon  a  mix- 
ture of  antitoxin  and  toxin  as  analogous  to  an  amphoteric  mixture  of  a  dilute  acid 
and  alkali,  or  of  an  acid  and  alcohol.  In  such  combinations  there  are  com- 
pounds formed  of  the  two  substances,  but  some  of  each  of  the  two  constituents 
remains  free.  An  ester  is  not  only  a  compound  formed  by  an  acid  and  an  alco- 
hol, but  it  has  free  alcohol  and  free  acid.  Moreover,  the  ester  is  constantly 
changing,  some  of  the  alcohol  and  some  of  the  acid  separating  and  new  ester 
constantly  forming  again.  When  first  mixed,  more  ester  is  formed,  and  less 
alcohol  and  acid  are  liberated,  till  a  point  of  dynamic  equilibrium  is  reached, 
when  just  as  much  ester  is  formed  as  there  are  alcohol  and  acid  liberated.  Just 
so  in  adding  toxin  to  antitoxin:  at  first  more  of  the  two  combine  than  is  set  free, 
but  after  a  time  a  condition  of  dynamic  equilibrium  is  established,  and  any 
additional  toxin  remains  free. 

Briefly  stated,  these  are  the  three  theories  which  are  now  advanced  by  com- 
petent authorities,  and,  if  these  outlines  are  kept  clearly  in  mind,  it  will  not  be 
difficult  to  understand  the  subject  as  presented  in  the  many  medical  journals 
and  the  many  monographs  which  have  appeared  on  the  subject.* 

The  ihsoihs  of  Ehrlich  and  Bordet  in  regard  to  the  composition  of  lysins 
may  also  be  appropriately  discussed  in  this  connection,  as  it  is  from  the  studies 
of  these  bodies  that  many  of  the  ideas  in  regard  to  immunity  have  been  developed. 

A  lysin  contains  two  substances,  a  ihermolabile  and  a  thermostabile  substance 
— i.  e.,  one  readily  destroyed  by  heating  at  55°  C.  for  a  half-hour,  the  other  resist- 
ing much  higher  temperatures.  The  thermostabile  substance  is  now  called  by 
Ehrlich  the  immune  body,  the  thermolabile  the  complement,  though  Ehrlich  has 
used  in  the  past  various  other  names  for  these  hypothetical  bodies.  Bordet  uses 
the  name  substance  sensibilisatrice  for  the  thermostabile,  Ehrlich's  immune  body, 
and  alexin  for  the  thermolabile  or  Ehrlich's  complement.  Both  are  agreed  that 


*A  summary  will  be  found  in  a  monograph  by  Michaelis.  Die  Bindungsgesetze 
von  Toxin  und  Antitoxin.  Berlin.  1905.  Also  a  special  article  entitled. 
Immunity.  Journal  of  the  American  Medical  Association.  Nos.  4  el  seq. 
1905. 


IMMUNITY.  227 

there  are  two  bodies  concerned:  both  are  agreed  as  to  the  property  of  the  one  to 
be  readily  destroyed  by  heat,  of  the  other  to  resist  heat. 

In  English  writing  it  is  more  common  to  use  the  German  than  the  French 
te  ms,  so  these  will  be  employed  in  the  present  case,  though  a  great  deal  of  what 
is  known  about  lysins  has  been  contributed  by  Bordet  and  the  French  school 
generally.  The  word  alexin  was  first  used  by  Buchner,  but  is  used  now  mostly 
in  French  writings. 

It  should  be  recalled  that  a  lysin  is  the  substance  formed  in  the  blood-serum 
of  an  animal  when  the  latter  is  injected  with  bacteria  or  with  foreign  red  blootl 
cells.  A  rabbit  injected  with  typhoid  bacilli  develops  lysin  for  typhoid  bacilli; 
when  injected  with  red  blood-cells  of  a  guinea-pig,  develops  a  lysin  for  guinea- 
pig  red  cells.  Lysins  are  not  only  produced  artificially  by  such  injections  bui 
they  may  also  be  present  in  blood-serum  normally. 

If  the  lysin  is  heated  to  55°  C.  for  thirty  minutes,  it  loses  its  complement  (or 
alexin}  and  the  immune  body  (substance  sensibilisatrice)  only  remains;  so  that 
red  cells  which  are  disintegrated  by  the  unhoated  lysin  remain  intact  in  the  heated 
lysin.  But  the  heated  lysin  becomes  active  again  if  either  fresh  unheated  rabbit's 
or  guinea-pig's  serum  is  added  to  it.  The  heated  lysin  is  spoken  of  as  inactivated- 
the  heated  lysin  with  fresh  serum,  reactivated.  The  fresh  serum  which  is  added 
contains  the  complement  (alexin);  the  heated  lysin  contains  only  the  immune 
body  (substance  sensibilisatrice). 

The  immune  body  is  specific,  but  the  complement  is  not;  at  least  the  blood  of 
Some  animals  contains  complements  for  several  different  immune  bodies.  Thus 
fresh  horse  serum  added  to  various  inactivated  lysins  reactivates  the  latter. 
But  chicken  blood-serum  does  not  contain  complement  for  chicken  corpuscles. 
For  if  chicken  hemolysin,  produced  by  injecting  a  rabbit  with  chicken  red  cells, 
is  heated  to  55°  C.  for  thirty  minutes  (inactivated),  it  will  not  disintegrate  chicken 
red  cells  if  fresh  chicken  serum  be  added;  but  if  fresh  rabbit  serum  is  added, 
it  will  hemolize  chicken  red  cells  as  it  did  before  heating. 

The  immune  body  becomes  fixed  to  the  red  cells,  as  can  be  shown  by  adding 
red  cells  to  inactivated  lysin  and  then  washing  these  with  salt  solution.  If  after 
adding  the  red  cells  to  the  inactivated  lysin  the  mixture  is  centrifugalized  and 
the  precipitated  red  cells  washed  with  salt  solution  so  as  to  remove  all  of  the  free 
immune  body,  the  precipitated,  washed  red  cells  disintegrate  when  fresh  comple- 
ment— i.  e.,  fresh  serum — is  added. 

From  these  and  other  considerations  Bordet  regards  lysin  as  composed  of  a 
specific  antibody,  sensibilisatrice  or  immune  body  of  Ehrlich,  on  the  one  hand 
and  of  a  cytolytic,  bacteriolytic,  hemolytic  alexin  proper  or  complement  of  Ehr- 
lich, on  the  other.  The  immune  body  is  specific,  but  it  does  not  cause  destruc- 
tion of  cells  by  itself:  it  does  so  only  in  conjunction  with  complement  or  alexin. 
Alexin  is  not  strictly  specific,  and  it  has  some  cytolytic  power,  as  seen  in  normal 
blood  independently  of  substance  sensibilisatrice.  But  its  power  is  greatly  en- 
hanced if  the  cells  acted  upon  are  first  sensitized  by  sensibilisatrice. 

The  reactions  found  by  Bordet  may  be  briefly  summarized  as  follows: 


228  MANUAL    OF    BACTERIOLOGY. 

Bacteria  or  other  cells  which  are  united  to  the  same  immune  body  or  sensibili- 
satrice  become  disintegrated  upon  the  addition  of  diverse  complements. 

In  any  one  cytolytic  serum  the  complement  for  bacteria  and  for  red  cells  is  one 
and  the  same.  The  fixation  of  the  immune  body  takes  place  by  the  stroma  of 
the  red  cells,  for  red  cells  washed  of  all  their  contents  so  that  only  the  empty  cap- 
sules are  left  fix  the  immune  body  just  as  well  as  unwashed  red  cells. 

Antilysin  combines  with  both  immune  body  and  complement. 

Since  antilysin  neutralizes  the  complement,  it  is  both  antihemolytic  and  anti 
bacteriolytic,  because  the  complement  for  both  of  these  is  the  same. 

Pfeiff er  was  the  first  to  describe  the  bacterioly tic  action  of  animal  fluids  on  bac- 
teria, and  the  reaction  is  called  Pfeiffer's  phenomenon.  Pfeiffer's  view  of  the 
nature  of  lysin  differs  from  those  of  Ehrlich  and  of  Bordet.  He  holds  that  lysin 
is  not  composed  of  two  bodies,  but  that  it  is  one  body  which  is  readily  changed 
into  an  active  and  passive  condition.  This  view  does  not  seem  to  have  found  as 
much  favor  as  that  of  Ehrlich  and  that  of  Bordet. 


But  among  those  who  have  accepted  more  or  less  completely 
the  Ehrlich  conception  of  the  nature  of  the  bodies  concerned 
in  bacteriolysis,  there  is  difference  of  opinion  as  to  the  mode 
of  action  of  the  complement.  Ehrlich  himself  says  in  regard 
to  the  matter  "that  one  will  not  go  amiss  if  he  assumes  with 
Pfeiffer  that  the  process  of  bacteriolysis  is  analogous  to  diges- 
tion, and  attributes  to  the  addiment  (complement)  the  char- 
acter of  a  digestive  ferment."  Gruber,*  on  the  contrary,  con- 
tends that  the  complement  does  not  act  like  a  ferment,  and 
that  it  is  erroneous  to  draw  any  analogy  between  the  comple- 
ment and  an  enzyme,  since  the  complement  is  entirely  used  up 
in  bacteriolysis,  whereas  in  the  process  of  fermentation,  as  is 
well  known,  the  ferment  is  not  used  up,  but  may  be  recovered 
after  the  action  is  ended  and  used  for  the  fermentation  of 
other  material. 

But  whatever  the  exact  mode  of  action  may  be,  it  is  evident 
from  what  has  just  been  said  that  both  the  Ehrlich  and  the 
Bordet  schools  attribute  bacteriolytic  action  proper  in  normal 
serum  to  a  substance  easily  changed  by  comparatively  low 
temperatures,  and  called,  respectively,  complement  and  alexin 

*Gruber.  Kolle  and  Wassermann's  Handbuch  der  pathogenen  Mikroor- 
ganismen. 


IMMUNITY.  229 

by  the  two  schools.  To  the  other  body  concerned  in  bacterio- 
lysis— the  amboceptor  of  Ehrlich  and  the  substance  sen- 
sibilisatrice  of  Bordet — is  assigned  by  the  former  the  role  of  a 
binding  link  between  the  complement  and  the  bacterium, 
while  by  the  latter  is  assigned  to  it  the  property  of  a  sensitizer 
or  of  a  mordant  as  in  dyeing.  In  the  one  case  the  bacterial 
cell  is  regarded  as  not  at  all  injured  or  otherwise  changed  by 
the  union  with  the  amboceptor;  in  the  other  case  it  is  the  opin- 
ion of  those  holding  this  view  that  the  cell  is  acted  upon  and 
changed  by  the  sensibilisatrice  in  such  a  way  that  the  alexin 
can  penetrate  it. 

Bordet*  summarizes  the  difference  between  his  theory  and 
that  of  Ehrlich  as  follows : 

According  to  Ehrlich  and  Morgenroth  the  specific  antibody  (sensibilisatrice) 
plays  the  role  of  an  actual  intermediary  (zwischenkorper,  amboceptor),  a  link 
of  union  attaching  itself  on  the  one  hand  to  the  cell,  on  the  other  to  the  alexin. 
In  other  words,  the  absorption  which  the  alexin  undergoes  in  the  presence  of  the 
sensitized  cell  is  not  due  to  an  affinity  manifested  by  the  cell  itself  to  this  substance. 
The  absorption  of  the  alexin  is  only  indirect;  the  cell  joins  itself  to  the  interme- 
diary substance,  which  is  itself  united  chemically  by  its  other  pole  to  the  alexin. 

Our  idea  of  the  phenomenon,  which  we  feel  we  are  justified  in  holding,  is  alto- 
gether different  from  this.  To  us  it  seems  that  the  sensibilisatrice  which  unites 
with  the  cell  modifies  this  in  a  way  which  permits  it  to  absorb  the  alexin  directly. 
The  action  of  the  sensibilisatrice  upon  the  cells  is  comparable  to  that  of  certain 
fixative  agents  or  mordants  which  confer  upon  certain  substances  (or  to  the  cell 
of  these  substances,  as  is  the  case  in  histological  technic)  the  power  of  absorbing 
colors  which  they  refuse  to  absorb  before  treatment.  *  *  *  It  is  to  be  clearly 
understood,  however,  that  when  we  speak  of  mordants  in  this  connection  we  do 
not  intend  to  apply  in  all  details  the  phenomena  of  dyeing  to  the  matter  at  pres- 
ent under  consideration ;  we  merely  mean  to  draw  a  comparison  which  will  serve 
to  make  our  idea  clearer.  The  hypothesis  which  we  wish  to  bring  out  in  relief 
is  that  in  the  presence  of  hemolytic  serum,  the  cell  becomes  capable  of  absorbing 
directly  the  alexin  by  means  of  its  own  proper  elective  affinity,  and  that  this  power 
is  due  to  a  change  caused  by  the  sensibilisatrice.  In  other  words,  we  do  not 
believe  that  one  is  forced  to  admit,  with  Ehrlich  and  Morgenroth,  that  the 
sensibilisatrice  itself  combines  with  the  alexin,  and  that  this  union  is  indispensable 
for  the  latter  substance  to  attack  the  cell. 

*Bordet,  Jules.  Sur  le  mode  d'action  de  serums  cytolytiques  et  sur  Punite 
de  1'alexine  dans  un  meme  serum.  Ann.  de  1'inst.  Past.,  T.  15,  no.  5,  303—318. 
Paris,  May  25,  1901. 


23° 


MANUAL    OF    BACTERIOLOGY. 


Bordet  furthermore  states  in  the  same  connection  that  he 
agrees  with  Buchner  in  regarding  the  alexin  for  blood-cells  and 
for  bacteria  as  identical — that  one  and  the  same  alexin  may 
attack  the  most  diverse  cells;  whereas  Neisser  and  others  of  the 
Ehrlich  school  hold  that  alexins  or  complements  are  different 
in  one  and  the  same  serum. 

While  it  is  evident  from  the  above  that  the  terms  amboceptor 
and  sensibilisatrice  are  used  to  designate  the  same  substance,  it 
is  scarcely  correct  to  use  them  interchangeably,  since  they  con- 
note somewhat  different  at- 
tributes in  the  body  to  which 
they  refer.  The  same  is  true 
of  the  terms  complement  and 
alexin,  though  to  a  less  degree. 
The  following  diagrams, 
obtained  from  various  sources 
and  modified  to  suit  the  pur- 
pose, will  serve  to  illustrate 
the  process  of  bacteriolysis 
according  to  the  view  of  the 
Ehrlich  school. 

Fig.    56    represents    in    its 
simplest  form  the  mechanism 

of  bacteriolysis  according  to  the  Ehrlich  hypothesis,  and  serves 
to  illustrate  the  process  sufficiently  for  present  purposes. 

In  the  diagram  the  bacteria  are  represented  by  the  parts 
marked  b,  the  amboceptors  by  those  marked  a,  and  the  com- 
plements by  those  marked  k.  In  No.  i  the  bacterium,  am- 
boceptor, and  complement  are  represented  as  just  on  the  point 
of  uniting.  No.  2  represents  the  bacterium  and  the  ambocep- 
tor united  and  the  complement  on  the  point  of  uniting  with  the 
unoccupied  end  of  the  amboceptor.  No.  3  represents  the  pro- 
cess of  uniting  of  bacterium,  amboceptor  and  complement  com- 
pleted; the  bacterium  in  this  case  would  undergo  bacteriolysis. 


FIG.  56. — Illustration  of  the  mechan- 
ism of  bacteriolysis  according  to  the 
Ehrlich  hypothesis. 


IMMUNITY.  231 

It  should  be  borne  in  mind  that  according  to  this  theory 
bacteriolysis  can  take  place  only  where  the  bacterium  becomes 
united  to  an  amboceptor  which  is  itself  united  with  a  comple- 
ment. A  bacterium  may  become  united  with  a  free  ambocep- 
tor— i.  e.,  an  amboceptor  which  is  not  united  with  a  comple- 
ment— but  the  bacterium  in  such  a  case  does  not  undergo 
bacteriolysis  unless  a  complement  subsequently  becomes  at- 
tached to  the  amboceptor.  The  complement  is  incapable  of 
uniting  directly  with  a  bacterium;  it  can  do  this  only  through 
the  intervention  of  the  amboceptor.  But  when  the  comple- 
ment becomes  linked  to  the  bacterium  by  means  of  the  am- 
boceptor, the  bacterium  becomes  broken  up  into  minute  gran- 
ules and  ultimately  disappears. 

The  bonds  by  which  the  amboceptor  attaches  itself  to  the 
bacterium  on  the  one  hand,  and  to  the  complement  on  the 
other  are  called  haptophor  groups  or  haptophors  (h),  and 
similarly  this  name  is  given  to  the  bonds  of  union  of  the  bac- 
teria and  of  the  complement.  The  amboceptor  thus  has  two 
haptophors,  one  by  means  of  which  it  attaches  itself  to  the 
bacterium,  the  cytophylic  haptophor,  and  one  by  means  of 
which  it  attaches  itself  to  the  complement,  the  complemen- 
tophylic  haptophor.  The  bacteria  probably  possess  each 
many  haptophors,  all  of  the  same  kind — i.  e.,  haptophors 
capable  of  uniting  with  amboceptors  of  the  same  kind — but 
for  the  sake  of  simplicity  the  bacterium  is  represented  in  the 
diagram  as  having  only  one  haptophor.  The  complement  has 
one  haptophor  group  and  one  so-called  toxophor  group  (/),  and 
it  is  by  means  of  the  latter  group  that  the  complement  acts 
upon  the  bacterium.  The  complement  may  be  deprived  of 
this  toxophor  group,  and  although  it  is  still  capable  of  uniting 
with  the  amboceptor  in  such  a  case,  it  can  no  longer  cause 
bacteriolysis.  This  loss  of  the  toxophor  group  is  caused  by 
heating,  and  is  also  occurs  spontaneously  in  the  serum  on 
standing.  Bacteria  subjected  to  the  action  of  heated  serum 


232  MANUAL    OF    BACTERIOLOGY. 

do  not  undergo  bacteriolysis,  but  become  fixed  to  the  ambocep- 
tors, and  the  amboceptors  become  united  to  the  haptophor 
group  of  the  complement  which  are  left  unaffected  by  the 
heating.  It  will  thus  be  readily  understood  why  bacteria 
treated  with  heated  immune  serum  are  subsequently  pro- 
tected from  bacteriolysis  even  when  unheated  immune  serum 
or  when  unheated  complement  is  added  to  them.  The  com- 
plementophylic  haptophor  of  the  amboceptor  is  in  such  a  case 
already  occupied  by  the  haptophors  of  the  heated  complement, 
which  has  in  this  way  become  deprived  of  its  toxophor  group. 

The  amboceptors  found  in  ordinary  normal  serum  are  either 
all  alike — and  in  this  case  they  must  possess  affinity  for  a  great 
many  different  kinds  of  bacteria — or  they  must  differ  from  one 
another;  and  in  this  case  there  must  evidently  be  a  great  many 
specific  amboceptors,  some  fitted  for  the  bacteriolysis  of  one 
species  of  bacterium,  some  for  others.  This  matter  seems  not 
yet  to  have  been  settled.  But  it  is  certain  that  the  injection 
of  an  animal  with  certain  bacteria  or  their  products  causes  the 
formation  of  a  large  number  of  specific  amboceptors;  that  is  to 
say,  of  amboceptors  having  affinity  only  for  the  kind  of  bacteria 
with  which  the  animal  is  injected.  Such  injections  seem  not 
to  increase  the  amount  of  complement. 

Complement  is  found  normally  in  the  serum,  that  of  some 
animals  possessing  more  than  that  of  others.  The  horse  ap- 
pears to  have  a  large  amount  of  complement  in  the  serum.  It 
is  not  yet  settled  whether  the  complement  is  specific — that  is, 
whether  the  complement  for  one  kind  of  immune  serum  can 
unite  with  the  amboceptors  of  this  serum  only  and  not  with 
the  immune  serum  of  a  different  sort — or  whether  comple- 
ments are  general;  though  they  seem  for  the  most  part  not  to 
be  specific.  The  complement  in  the  serum  of  horse's  blood 
seems  capable  of  reactivating  heated  immune  serum  of  various 
kinds.  Still  in  some  cases  it  would  appear  as  if  they  were 
specific. 


IMMUNITY. 


233 


With  the  explanation  given  above  of  the  nature  of  ambocep- 
tors  and  complements,  the  phenomena  which  take  place  in  im- 
mune serum  become  more  or  less  satisfactorily  explicable.  By 
means  of  the  characteristics  ascribed  to  these  bodies  it  is  pos- 
sible to  account  for  the  peculiar  behavior  of  immune  serum 
when  it  is  diluted.  This  peculiar  behavior  consists  in  the 
fact  that  such  serum  is  frequently  more  potent  when  diluted 
than  when  it  is  undiluted. 

Neisser  and  Wechsberg  were  the  first  to  offer  a  theoretical 
explanation  of  this  phenomenon,  and  they  very  appropriately 
call  their  explanation  the  theory  of  the  diversion  of  complement. 


FIG.  57. — Diversion  of  complement  in  undiluted  immune  serum. 

As  the  name  implies,  they  attribute  the  lack  of  bacteriolysis 
in  the  undiluted  immune  serum  to  the  turning  aside  of  the 
complement  from  the  bacteria,  or  rather  from  the  amboceptors 
which  are  attached  to  the  bacteria.  They  hold  that  this 
diversion  is  brought  about  by  the  free  amboceptors  them- 
selves. In  other  words,  where  there  are  more  amboceptors. 
than  there  are  complements  present  in  a  serum,  a  part  of 
these  attach  themselves  to  the  bacteria  and  a  part  to  the 
complements. 

The  accompanying  diagram  (Fig.  57),  taken  from  Neisser 
and  Wechsberg,  and  modified  to  suit  the  present  description, 
shows  two  amboceptors,  a,  attached  to  bacteria,  b,  and  four 


234 


MANUAL    OF    BACTERIOLOGY. 


amboceptors  attached  to  complements,  k.  Bacteriolysis  is  not 
possible  in  such  a  condition,  because  the  complements  have 
been  diverted  from  the  amboceptors  which  are  attached  to  the 
bacteria.  Bacteriolysis  can  take  place  only  when  the  comple- 
ment becomes  attached  to  the  bacterium  through  the  medium 
of  the  amboceptor. 

Fig.  58  is  meant  to  show  the  same  serum  diluted  with  an 
equal  amount  of  salt  solution.  In  this  case,  with  the  same 
number  of  bacteria  added,  it  is  evident  that  one-half  of  them 
would  be  killed,  as  is  indicated  by  the  combination  between 

bacteria,  amboceptor,  and 
complement  in  Fig.  56.  The 
other  half  of  the  bacteria 
would  evidently  escape,  the 
complement  being  diverted  by 
the  free  amboceptor,  as  shown 

by  Figs-  57  and  58- 

The  conditions  under  which 
bacteriolysis  would  take  place 
and  those  under  which  no  bac- 
teriolysis would  take  place  in 
any  given  serum,  may  be  sum- 
marized as  follows : 
i.  Complete  bacteriolysis  could  take  place  only  where  there 
were  no  free  amboceptors  and  where  there  were  at  the  same 
time  a  number  of  amboceptor-complements  equal  to  or  greater 
than  the  number  of  bacteria  introduced. 

On  dilution  in  a  serum  of  this  kind  there  would  be  a  loss  of 
bacteriolytic  power  in  proportion  to  the  degree  of  dilution  if  the 
amboceptor-complements  were  exactly  equal  in  number  to  the 
bacteria  introduced.  If  there  were  more  amboceptor-com- 
plements originally  in  the  undiluted  serum  than  the  bacteria 
introduced,  then  on  dilution  there  would  be  relatively  more 
bacteria  destroyed.  If  the  excess  of  amboceptor-complements 


FIG.  58. — Partial  bacteriolysis  and 
partial  diversion  of  complement  in 
diluted  immune  serum. 


IMMUNITY.  235 

is  large  enough,  there  could  of  course  be  enough  present  in  the 
diluted  serum  to  kill  as  many  bacteria  as  were  killed  by  the 
serum  before  dilution. 

2.  Partial  bacteriolysis  would  follow  when  there  were  fewer 
amboceptors  present  in  the  serum  than  the  bacteria  introduced 
and  when  at  the  same  time  there  were. any  amboceptor-com- 
plements  present. 

The  extent  of  bacteriolysis  upon  dilution  would  depend 
upon  the  number  of  amboceptor-complements  present  orig- 
inally. 

3.  No  bacteriolysis  could  take  place  if  the  free  amboceptors 
were  equal  in  number  to  the  bacteria  introduced,  or  if  they 
were  in  excess  of  this  number,  either  in  the  undiluted  or  the 
diluted  serum. 

In  the  above  discussion  it  is  assumed  that  the  two  bodies 
concerned — the  amboceptor  and  the  complement  of  Ehrlich, 
the  sensibilisatrice  and  the  alexin  of  Bordet — are  capable  of 
uniting  and  do  actually  unite  independently  of  the  presence  of 
bacteria  or  of  other  cells.  But  Bordet*  nevertheless  published 
a  series  of  investigations  tending  to  show  that  the  experiments 
of  Ehrlich  and  Sachs,  which  constitute  the  chief  evidence  in 
favor  of  this  view,  are  capable  of  quite  a  different  interpreta- 
tion from  this,  and  that  this  interpretation  is,  in  fact,  not  justi- 
fiable from  the  results  of  the  experiments  which  consisted  in  the 
demonstration  of  the  fact,  not  denied  by  Bordet,  that  ox  serum 
will  produce  cytolysis  only  when  the  serum  has  in  it  ambocep- 
tors and  complements  simultaneously.  It  is  not  possible,  as  in 
some  other  cases,  to  produce  cytolysis  by  sensitizing  cells  with 
ox  amboceptors— that  is,  with  heated  ox  serum — and,  after 
washing  these  sensitized  cells,  adding  complement — that  is, 
fresh  serum.  Cytolysis  with- ox  serum  takes  place  only  when 

*BORDET,  JULES,  and  GAY,  FREDERICK  P.  Sur  les  relations  des  sensibil- 
isatrices  avec  1'alexine.  Ann.  de  Vlnst.  Past.,  t.  20,  No.  6,  p.  267-498.  Paris, 
June  25,  1906. 


236  MANUAL   OF    BACTERIOLOGY. 

the  heated  ox  serum  and  some  unheated  fresh  serum  (horse 
serum  was  the  kind  used  in  the  experiments)  are  employed  at 
the  same  time.  This  is  interpreted  by  Ehrlich  and  Sachs  as 
showing  that  while  the  free  amboceptors  present  in  the  ox 
serum  will  not  unite  with  the  cells  they  will  and  do  so  unite 
when  they  are  previously  attached  to  complements.  But 
Bordet's  results  appear  to  show  quite  plainly  that  in  this  case 
the  horse  serum  which  was  used  as  complement  produces 
cytolysis  quite  independently  of  the  ox  serum,  and  that  while 
cytolysis  takes  place  more  promptly  when  heated  ox  serum- 
ox  amboceptors — are  added,  the  ox  serum  is  not  necessary. 
Bordet  therefore  regards  the  experiments  as  showing  that  the 
heated  ox  serum  acted  merely  as^an  accelerator  of  cytolysis. 
Bordet  summarizes  his  conclusions  as  follows: 

We  see  but  one  rational  explanation  of  the  peculiar  action  of  ox  serum — 
that  there  exists  in  the  serum  a  peculiar  substance  capable  of  resisting  heat  of 
56°  C.  and  which  remains  unaltered  for  many  months  in  this  heated  serum. 
The  substance  is  probably  of  an  albuminous  or  colloid  character,  and  does  not 
adhere  to  the  normal  corpuscles,  but  is  precipitated  upon  the  corpuscles  which 
are  previously  charged  with  sensibilisatrice  and  alexin.  We  believe  that  it  is 
a  veritable  process  of  glueing  or  absorption  depending  upon  molecular  adhesion 
*  *  *  In  conformity  with  the"  statements  of  Ehrlich  and  Sachs,  experiments 
show  that  the  corpuscles  of  guinea-pigs  become  hemolyzed  in  a  mixture  of  fresh 
serum  and  of  ox  serum,  the  latter  having  been  heated  to  56°  C.,  while  they  resist 
hemolysis  if  they  are  first  subjected  to  the  action  of  the  heated  ox  serum  and  have 
the  horse  serum  added  subsequently.  But  the  interpretation  offered  by  Ehrlich 
and  Sachs,  according  to  which  the  sensibilisatrice  furnished  by  the  ox  serum 
does  not  unite  with  the  corpuscles  unless  it  (the  sensibilisatrice)  is  previously 
connected  with  alexin  derived  from  the  horse  serum  it  not  correct.  In  the  first 
place,  the  sensibilastrice,  which  plays  a  preponderating  and  most  essential  role 
is  not  contained  in  the  ox  serum  at  all,  but  is  furnished  by  the  horse  serum. 
Consequently  these  sensibilisatrices  behave  like  all  of  their  congeners,  in  the 
sense  that  they  do  not  require  the  presence  of  the  alexin  before  they  are  capable 
of  uniting  with  the  corpuscles. 

Finally,  this  interpretation  leaves  completely  in  the  dark  the  very  special 
peculiarities  of  the  cases  of  hemolysis  in  question. 

The  peculiarity  of  ox  serum  consists  in  the  presence  of  a  certain  element  which 
resists  56°  C.,  and  also  resists  standing,  and  is  of  the  nature  of  a  colloid,  doubtless 
albuminoid,  and  which,  furthermore,  is  absorbed  by  corpuscles  which  have  become 


IMMUNITY.  237 

charged  with  sensibilisatrice  and  alexin  but  which  remains  free  in  the  presence 
of  normal  corpuscles  or  of  corpuscles  merely  sensitized — i.  c.,  corpuscles  treated 
with  heated  serum  alone.  The  absorption  of  this  colloid  by  corpuscles  which 
have  been  treated  with  both  sensibilisatrice  and  alexin  has  the  effect  of  enegeti- 
cally  agglutinating  them  and  of  rendering  them  more  susceptible  to  hemolysis 
except  under  certain  circumstances.  *  *  *  The  absorption  of  the  sensitized 
and  alexinized  corpuscles  is  very  likely  due  to  molecular  adhesion,  the  prelimi- 
nary treatment  having  modified  the  corpuscles  in  so  far  as  their  adhesive  proper- 
ties are  concerned.  Under  these  conditions  the  absorption  may  take  place 
independently  of  the  species  of  animal  from  which  the  corpuscles  are  obtained, 
it  may  even  take  place  with  the  corpuscles  of  the  same  animal  which  furnishes 
the  colloid,  as  in  the  case  of  ox  serum. 

Now,  if  the  contention  of  Bordet  as  set  forth  above  is  cor- 
rect, and  if  the  two  bodies  concerned  in  cytolysis  do  not  unite, 
then  of  course  the  theory  of  complement  diversion  must  fall, 
since  it  requires  as  the  first  condition  the  union  of  amboceptors 
with  complements.  Nevertheless,  there  appears  to  be  as  yet 
no  other  explanation  of  the  phenomena  observed  on  diluting 
immune  serum- 

While  it  is  true  that  immune  blood-serum  differs  from  normal 
blood-serum  in  the  matter  of  its  behavior  on  dilution,  this  differ- 
ence is  one  of  degree  rather  than  of  kind.  For  normal  blood- 
serum  kills  relatively  more  bacteria  when  it  is  diluted  than  when 
undiluted.*  Normal  serum  which  will  kill  a  given  number  of 
bacteria  when  undiluted  will  kill  a  great  many  more  than  y1-^ 
that  number  when  diluted  1:10;  it  will  kill  a  great  many  more 
than  T-j3"o  of  the  number  killed  by  the  undiluted  serum  when 
diluted  in  the  proportion  of  i  :iooo.  In  some  cases  the  normal 
serum  will  not  only  kill  relatively  more  bacteria  than  the 
undiluted,  but  actually  more. 

*Bolton.  The  Bacteriolytic  Power  of  the  Blood-serum  of  Hogs.  U.  S.  De- 
partment of  Agriculture.  Bu.  An.  In.  Bulletin  No.  95. 


CHAPTER  VII. 
DISINFECTION,   STERILIZATION   AND   ANTISEPSIS. 

THE  means  employed  for  the  destruction  of  the  bacteria  and 
for  the  prevention  of  their  growth  are  of  interest  in  several 
directions:  in  the  prevention  of  the  spread  of  infectious  dis- 
eases in  the  avoidance  of  the  infection  of  wounds,  both  acci- 
dental wounds  and  those  produced  in  operative  proceedures; 
and  in  the  preservation  of  food-stuffs  and  other  perishable 
material. 

Disinfection  is  the  term  employed  to  signify  the  destruction  of 
the  infectious  agent  in  the  rooms,  stables,  barns,  cars  or  other 
ocnfined  spaces  previously  occupied  by  persons  or  animals  suf- 
fering from  an  infectious  disease,  also  the  furniture  in  the  rooms 
and  the  clothing  worn  by  such  persons.  It  is  also  used  for 
the  process  employed  destroying  the  infectious  agent  in  feces, 
urine,  sputa  and  other  excreta  from  persons  suffering  from  in- 
fectious disease.  The  term  always  implies  the  presence  of  an 
infectious  agent  which  is  to  be  got  rid  of,  it  is  better  to  avoid 
using  the  term  for  processes  employed  in  masking  or  destroy- 
ing disagreeable  odors  arising  from  improperly  flushed  water- 
closets  or  decaying  offal. 

Sterilization  is  used  to  denote  the  destruction  of  bacteria  in 
general.  It  does  not  necessarily  imply  the  destruction  of 
pathogenic  bacteria.  Thus  in  the  preparation  of  culture 
media,  the  vessels  and  the  media  themselves  are  sterilized  by 
means  of  heat,  although  there  may  or  may  not  be  pathogenic 
bacteria  in  them  to  start  with. 

Antisepsis  is  the  term  applied  to  the  process  of  preventing  the 
growth  of  bacteria  in  wounds  or  in  articles  of  food  or  elsewhere, 

238 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          239 

in  other  words,  to  the  prevention  of  putrefaction,  fermentation 
and  wound  infection.  Although  in  practice  antisepsis  usually 
involves  the  destruction  of  bacteria  as  well  the  prevention  of 
their  growth,  it  does  not  necessarily  involve  sterilization.  Cold 
prevents  the  growth  of  bacteria,  and  therefore  acts  as  an  anti- 
septic, and  yet  it  does  not  act  as  a  germicide,  as  will  be  shown 
later.  Chloroform  added  to  urine  acts  as  an  antiseptic,  but 
it  does  not  kill  the  bacteria,  for  after  the  chloroform  is  evapo- 
rated off  the  bacteria  grow  out  in  the  urine. 

The  destruction  of  bacteria  and  the  prevention  of  their 
growth  is  accomplished  by  both  chemical  and  physical  agents. 
The  chemical  agents  are  some  of  them  applicable  in  aqueous 
solution,  and  some  of  them  as  gases.  All  agents  which  kill 
bacteria  are  called  germicides,  but  their  action  depends  upon 
the  intensity  and  the  length  of  time  they  are  allowed  to  act. 
If  they  are  used  in  dilute  solutions  or  if  they  are  allowed  to  act 
for  a  short  time  they  may  rapidly  inhibit  the  growth  or  produce 
some  change  in  the  bacteria  affecting  the  physiological  activity 
of  the  organisms.  Various  permanent  modifications  have 
been  produced  in  certain  bacteria  by  the  action  of  germicides 
applied  in  such  a  way  as  not  to  destroy  the  organisms  outright. 
Thus  the  anthrax  bacillus  has  been  deprived  of  its  power 
to  form  spores,  it  has  also  been  made  to  assume  graded  viru- 
lence so  that  it  would  kill  mice  but  not  sheep.  These  modi- 
fications are  produced  by  agencies  which  would  destroy  the 
bacterium  of  anthrax  if  allowed  to  act  with  sufficient  intensity. 

Germicides  are  all  much  more  active  when  dissolved  in  water 
than  when  dissolved  in  any  other  solvent,  though  it  is  true  that 
Epstein  found  that  bichloride  of  mercury,  carbolic  acid,  lysol 
and  thymol  were  more  powerful  when  dissolved  in  50  per  cent, 
alcohol  than  when  dissolved  in  the  same  proportion  in  water, 
though  authorities  differ  on  this  point.  Solutions  in  oils  are 
inert.  The  reason  for  this  is  that  the  bacterial  cell  is  pene- 
trated only  by  water,  not  by  oil.  The  addition  of  alcohol  to 


240  MANUAL    OF    BACTERIOLOGY. 

phenol  or  to  formaldehyde  lessens  the  germicidal  property  of 
these  germicides.  The  addition  of  even  5  per  cent,  of  alcohol 
shows  this  effect.  On  the  other  hand,  the  addition  of  alcohol 
to  aqueous  solutions  of  corrosive  sublimate  or  silver  nitrate 
increases  the  germicidal  power  of  these  germicides.  The 
addition  of  glycerin  to  solutions  of  germicides  acts  irregularly, 
increasing  the  power  in  some  of  them,  decreasing  the  power  in 
others. 

Researches  in  physical  chemistry  have  shown  that  the  disin- 
fecting power  of  metalic  salts  is  in  proportion  to  their  electro- 
lytic dissociation,  the  more  strongly  dissociated  a  salt  is  by 
electrolysis,  the  stronger  its  disinfecting  power. 

Scheurlen  and  Spiro  have  shown  this  with  iron  salts.  From 
their  results  it  appears  that  only  those  salts  of  iron  act  as 
germicides  in  which  the  iron  is  present  as  kation.  On  the 
other  hand,  all  disinfectants  do  not  act  in  this  way.  Phenol 
and  its  derivatives  do  not  act  through  the  ions.  In  this  case 
it  is  precisely  those  molecules  which  are  not  dissociated  which 
act  as  germicides.  Sodium  phenolate  is  much  more  strongly 
dissociated  than  phenol  itself,  and  yet  phenol  is  much  more 
strongly  germicidal  than  the  salt.  But  with  the  metalic  salts 
anything  which  interferes  with  their  dissociation  weakens 
their  disinfecting  power.  The  addition  of  sodium  chloride 
interferes  with  the  dissociation  of  corrosive  sublimate  and 
weakens  its  germicidal  power.  This  is  shown  more  strongly 
in  concentrated  than  in  dilute  solutions.  In  the  dilute  solu- 
tions of  corrosive  sublimate  ordinarily  employed  in  practice  the 
addition  of  the  salt  does  not  weaken  the  germicidal  power 
very  markedly. 

Temperature  has  a  marked  effect  upon  the  action  qf  ger- 
micidal agents.  The  higher  the  temperature,  the  more  potent 
the  germical  action,  but  just  the  reverse  is  true  in  regard  to 
the  inhibitory  action  of  germicides.  The  inhibitory  action  is 
weakest  at  the  optimum  temperature  for  the  growth  of  the  bac- 


DISINFECTION,    STERILIZATION    AND   ANTISEPSIS.          241 

terium  under  observation.  It  is  stronger  in  the  cold.  The  ex- 
planation offered  for  this  behavior  is  that  the  vigor  of  growth 
at  the  optimum  temperature  overcomes  the  inhibitory  action 
of  the  germicide,  whereas  at  lower  temperatures  the  vigor  of 
the  organism  is  lowered,  and  therefore  it  is  less  capable  of 
resisting  the  inhibitory  action  than  at  the  more  suitable  tem- 
perature for  growth. 

The  chemical  composition  of  the  medium  in  which  the 
bacteria  are  tested  may  have  a  marked  influence  upon  the 
action  of  germicides.  If  components  of  the  medium  enter  into 
chemical  union  with  the  germicide  there  may  be  an  inert 
compound  formed.  There  may  also  be  formed  dense,  floccu- 
lent  precipitates  which  envelop  the  bacteria  and  protect  them 
from  the  action  of  the  germicide.  It  is  therefore  apparent  that 
the  potency  of  a  germicide  may  appear  very  different  when 
acting  upon  the  bacteria  in  water  or  in  physiological  solution 
or  on  bacteria  dried  on  glass  rods  or  on  silk  threads,  on  the 
one  hand,  and  upon  the  same  bacteria  in  beef-broth  or  in  feces 
or  in  urine,  on  the  other.  For  these  reasons  it  is  not  always 
possible  to  draw  conclusions  from  the  results  of  laboratory 
experiments  as  to  the  value  of  a  germicidal  agent  for  practical 
disinfecting  purposes. 

The  action  of  germicides  is  still  further  complicated  by  the 
difference  of  resistance  shown  not  only  by  different  species  of 
bacteria,  but  by  the  different  strains  of  the  same  bacterium  and 
even  by  different  individuals  in  the  same  culture.  Further- 
more, some  bacteria  are  more  sensitive  than  others  to  certain 
chemical  agents.  In  other  words,  some  bacteria  have  an  elec- 
tive affinity  for  certain  chemicals  while  others  have  greater 
affinity  for  other  chemical  agents.  As  examples  of  this  elec- 
tive affinity  of  certain  bacteria  for  certain  germicides,  Got- 
schlich*  cites  the  action  of  quinine  in  malaria,  the  mercury 
salts  in  syphilis,  and  the  specific  action  of  bactericidal  sera. 

*Loc.  cit. 
16 


242  MANUAL    OF    BACTERIOLOGY. 

The  many  conditions  affecting  the  action  of  germicidal  agents 
probably  account  for  the  greater  or  less  discrepancy  in  the 
results  obtained  by  various  investigators  in  testing  the  value  of 
disinfectants.  Moreover,  it  has  been  shown  that  the  failure  of 
the  bacteria  to  grow  after  exposure  to  a  germicide  cannot  be 
regarded  as  a  certain  criterion  of  the  death  of  the  organism 
as  is  usually  done;  for  enough  of  the  germicide  may  be  trans- 
ferred in  the  inoculation  to  inhibit  growth  without  actually 
killing  the  bacteria.  Enough  of  the  germicide  may  even  ad- 
here to  bodies  of  the  bacteria  to  inhibit  growth  and  yet  not  kill 
the  bacteria,  as  shown  by  ultimate  growth  or  by  growth  after 
neutralizing  the  chemical  agent.  In  all  tests  of  the  germicidal 
action  of  chemical  agents  it  is  necessary  to  exclude  this  source 
of  error.  The  bacteria  should  be  thoroughly  washed  after 
exposure  to  the  germicide  and  if  possible  treated  with  some 
neutralizing  agent  which  is  not  itself  a  germicide  and  which 
does  not  form  a  germicidal  compound  with  the  germicide  which 
is  being  tested.  The  culture  tubes  or  plates  made  to  test  the 
viability  of  the  organisms  after  exposure  must  be  observed  for 
a  much  longer  time  than  is  done  with  ordinary  cultures;  for 
development  may  be  delayed  for  many  days  and  yet  take  place 
abundantly  at  last. 

The  manner  in  which  disinfectants  destroy  bacteria  differs 
in  different  cases.  In  some  cases  the  injurious  effect  is  due 
to  interference  with  the  nutrition,  in  some  cases  it  is  due  to 
oxidation;  but  much  more  frequently  it  is  due  to  coagulation  of 
the  protoplasm  of  the  cell.  Heat,  metalic  salts,  phenol,  all 
act  in  this  way.  On  the  other  hand,  not  every  agent  which 
coagulates  albumin  acts  as  a  powerful  germicide.  Alcohol 
and  tanic  acid  are  weak  disinfectants,  and  yet  they  coagulate 
albumin  very. strongly.  Lysins  found  in  blood-serum  act  by 
breaking  up  and  disintegrating  the  bacteria. 

The  methods  employed  for  testing  the  germicidal  value 
of  any  disinfectant  are  as  follows: 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          243 

1.  To  a  measured  quantity  of  a  virulent  bouillon-culture 
of  the  test-organism  is  added  a  given  amount  of  the  germi- 
cide.    After  varying  lengths  of  time,  inoculations  are  made  from 
this  mixture  into  culture-media,  preferably  bouillon,  and  note 
is  made  of  the  presence  or  absence  of  growth  under  suitable 
conditions  of  temperature  and  the  like.     The  shortest  exposure 
to  the  weakest  solution  of  the  substance  necessary  to  kill  the 
test-organism  is  taken  as  the  germicidal  value  of  that  substance 
for  the  particular  organism  used. 

2.  Small  pieces  of  sterile  silk  or  cotton  thread  are  soaked 
for  some  hours  in  a  bouillon-culture  of  the  test-organisms. 
The  threads  are  then  removed,  partially  dried  and  placed  in  a 
solution  of  known  strength  of  the  germicide  and  exposed  for  a 
definite  length  of  time.     The  thread  is  removed  from  the  solu- 
tion, washed  carefully  in  sterile  water,  dropped  into  a  tube  of 
sterile  bouillon  plugged  with  cotton,  and  growth  or  absence 
of  growth  noted.     As  in  other  methods,  the  greatest  dilution 
of  the  germicide  that  will  kill  the  test-organism  in  the  shortest 
time  is  taken  as  the  germicidal  value  of  that  substance  for  the 
organism  used.     Spores  of  the  hay  bacillus  may  be  used  when 
experiments  are  being  made  by  large  classes  of  students. 

3.  The    test-organism  is  dried   upon    the  end  of  a  sterile 
glass  rod  contained  in  a  sterile  test-tube,  the  end  of  the  rod 
projecting  through  a  cotton  plug.     The  end  of  a  glass  rod  is 
immersed  in  a  fluid  culture  of  the  test-organism  and  allowed 
to  dry.     While  drying  it  is  inserted  into  a  sterile  test-tube, 
and  plugged  around  with  cotton.     It  is  then  ready  to  test  by 
exposure  to  any  germicide,  either  liquid  or  gaseous.     After 
exposure  to  the  germicide  it  is  plunged  into  a  tube  of  sterile  beef- 
broth  in  order  to  see  whether  the  organisms  adhering  are  all 
killed. 

All  of  these  methods  are  open  to  serious  sources  of  error,  par- 
ticularly in  the  testing  of  powerful  germicides.  In  method  No.  i, 
small  quantities  of  the  substances  tested  may  be  carried  over  with 


244  MANUAL    OF    BACTERIOLOGY. 

the  organisms,  and,  if  a  powerful  germicide,  in  sufficient 
amount  to  prevent  growth,  and  thus  give  erroneous  results. 
In  methods  Nos.  2  and  3  this  factor  may  be  partially  obviated 
by  washing  in  sterile  water  after  exposure  to  the  germicide. 
This  does  not  remove  another  source  of  error,  namely,  the 
chemical  action  that  may  take  place  between  the  substance  and 
the  protoplasmic  contents  of  the  bacterial  cell.  This  action 
may  extend  deeply  enough  to  restrain  the  growth  of  an  organ- 
ism for  a  very  long  time  without  actually  killing  it.  When 
placed  under  suitable  conditions,  such  union  may  be  broken 
up  and  the  organism  regain  its  power  to  develop.  It  has  been 
suggested  that,  to  remove  errors  in  the  above  methods,  the 
bacteria  after  exposure  to  the  germicide  be  inoculated  into 
susceptible  animals;  but  Sternberg's  experiments  in  this  direc- 
tion have  shown  that  bacteria  may  become  so  attenuated  in 
virulence  by  the  action  of  germicides  insufficient  to  kill  that 
the  value  of  animal  inoculation  experiments  is  limited.  More- 
over, it  sometimes  happens  that  it  is  desired  to  test  germicides 
on  bacteria  which  are  not  pathogenic  for  animals. 

Geppert  suggested  a  valuable  modification  of  these  methods 
while  determining  the  germicidal  value  of  bichloride  of  mer- 
cury. After  exposing  his  test-organism  to  bichloride  of  mer- 
cury, and  before  inoculating  into  bouillon  to  determine  death 
of  the  organism,  he  treated  with  a  dilute  ammonium  sulphide 
solution,  thus  effectually  neutralizing  any  mercury-salt 
remaining. 

Sedgwick  developed  this  method  still  further,  and  to  him 
we  are  indebted  for  demonstrating  its  accuracy  and  practica- 
bility. Sedgwick  proceeds  as  follows: 

4.  To  15  c.c.  of  sterile  water  in  a  60  c.c.  Erlenmeyer  flask 
add  2  c.c.  of  a  virulent  culture  of  the  test-organism.     Then  add 
a  solution  of  the  substance  under  investigation  in  the  propor- 
tion necessary  to  give  the  dilution  wished.     Mix  thoroughly, 
and  allow  this  "action-flask"  to  stand  as  long  as  it  is  desired  to 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          245 

have  the  germicide  in  contact  with  the  test-organism  (action- 
period).  Transfer  0.5  c.c.  from  the  action-flask  to  a  flask 
containing  200  c.c.  of  a  solution  of  some  chemical  capable  of 
decomposing  the  substance  being  tested  with  the  formation  of 
inert  or  insoluble  compounds.  In  this  "inhibition-flask"  the 
strength  of  the  solution  should  be  such  that  molecular  propor- 
tions of  the  chemical  are  present  in  sufficient  quantity  to  com- 
bine with  all  the  germicides  carried  over.  The  inhibition-flask 
is  shaken  for  thirty  seconds,  and  i  c.c.  transferred  from  it  to  100 
c.c.  of  sterile  water  in  another,  the-"  dilution-flask."  After  two 
minutes,  three  agar  tubes  are  inoculated  with  i  c.c.  each  from 
the  dilution-flask,  plated,  and  growth  watched  for. 

Control-experiments  should  be  performed  to  determine  that 
the  dilution  of  the  test-culture  is  not  too  great  when  carried 
through  the  three  flasks.  It  likewise  should  be  determined  that 
the  inhibiting  chemical  itself  has  no  injurious  effect  on  the 
bacteria. 

The  inhibiting  chemical  must  be  determined  for  each  in- 
dividual case.  For  salts  of  the  heavy  metals ,  ammonium 
sulphide  answers  well;  for  mercury-salts,  stannous  chloride 
may  be  used;  for  formaldehyde,  ammonium  hydrate;  for  car- 
bolic acid,  sodium  sulphate. 

The  testing  of  gaseous  disinfectants,  such  as  sulphur  dioxid 
and  formaldehyde,  should  be  conducted  under  conditions  as 
nearly  identical  with  those  met  with  in  actual  practice  as  pos- 
sible. The  test-organisms  may  be  exposed  on  threads  or 
glass  rods,  and  acted  upon  by  a  known  volume  of  strength  of 
•germicide  for  a  known  length  of  time.  Subsequent  treatment 
of  the  organisms  with  a  suitable  inhibitor  is  necessary  when 
possible  and  should  growth  occur  in  the  cultures  following,  the 
test-organism  should  be  identified  in  order  to  exclude  possible 
contamination  by  extraneous  organisms. 

In  determining  the  value  of  germicides  for  sterilizing  liga- 
tures, the  students  can  apply  methods  based  on  the  foregoing 


246  MANUAL    OF    BACTERIOLOGY. 

principles.  Great  care  is  necessary  to  arrive  at  correct  conclu- 
sions, particularly  in  the  case  of  animal  tendons.  In  many  in- 
stances quite  stable  compounds  are  formed  between  tendon  and 
germicide,  and  living  organisms  may  be  so  imbedded  in  such 
a  substance  that  subsequent  growth  in  a  test-cuture  is  impos- 
sible. The  use  of  a  suitable  inhibitor,  and,  prior  to  final 
culture-tests,  a  prolonged  soaking  in  sterile  water,  will  promote 
the  accuracy  of  the  results. 

So  many  and  often  such  obscure  chemical  and  physical  fac- 
tors enter  into  the  action  of  chemical  germicides  that  uniform 
results  are  not  possible  within  narrow  limits.  This  accounts 
for  the  conflicting  results  obtained  by  different  investigators, 
and  even  the  same  investigator  at  different  times.  A  number 
of  variable  and  only  partially  controllable  conditions  enter  into 
every  test.  Results  with  gaseous  disinfectants  are  especially 
uncertain  on  this  account.  No  gaseous  disinfectants  have 
any  great  power  of  penetration,  and  consequently  act  only 
where  the  bacteria  are  freely  exposed  and  then  not  always 
with  certainty. 

Physical  Disinfectants.—  Drying.—  The  effect  of  drying 
differs  with  different  organisms  and  also  with  the  same  organ- 
ism depending  upon  whether  there  are  spores  present  or  not. 
The  vegetative  cells  of  some  organisms  are  very  readily  killed 
by  drying,  but  spores,  on  the  contrary,  are  not  affected  in  this 
way.  Spores  have,  in  fact,  been  preserved  for  years  dried  on 
silk  threads. 

Agitation.— "Bacteria,  are  killed  by  prolonged  violent  agita- 
tion. They  may  be  actually  shaken  to  pieces.  But  ordinary 
gentle  shaking  appears  in  some  cases  at  least  to  stimulate 
growth. 

Cold.—  Bacteria  are  very  resistant  to  cold,  those  that  have 
been  so  tested  survive  the  low  temperature  of  liquid  air, 
though  of  course  there  is  no  growth  at  this  temperature.  The 
organisms  subjected  to  this  low  temperature  grow  readily 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          247 

when  inoculated  upon  culture  media  and  removed  to  a  suit- 
able temperature. 

Light. — Light  is  injurious  to  bacteria.  Even  diffuse  day 
light  is  harmful,  and  direct  sun  light  is  distructive.  Electric 
arc  light  is  also  injurious.  The  rays  which  are  active  are  those 
at  the  ultra-violet  end  of  the  spectrum. 

Heat. — Bacteria  are  very  sensitive  to  heat.  Even  tempera- 
tures which  are  far  short  of  burning  or  charring  kill  the  bac- 
teria very  promptly.  The  vegetative  cells  are  for  the  most  part 
destroyed  by  temperatures  around  60°  C.  in  a  comparatively 
short  time, — ten  or  fifteen  minutes.  Spores  are  much  more 
resistant  to  heat,  some  of  these  withstand  the  temperature  of 
boiling  water  for  a  few  minutes  and  some  even  for  an  hour 
or  more. 

Other  Physical  Agents. — Pressure,  Rontgen  rays,  electric 
currents  are  not  injurious  to  the  bacteria. 

CHEMICAL  DISINFECTANTS. 

The  most  efficacious  method  of  disinfection  is  by  heat  applied 
as  steam  preferably  under  pressure.  This  mode  of  disinfec- 
tion is  applicable  to  bed  clothing  and  underclothing,  but  it  is 
not  applicable  to  rooms  and  the  like,  and  for  this  purpose 
recourse  is  had  to  various  chemical  agents.  The  list  of  sub- 
stances possessing  germicidal  properties  is  large,  but  not  all  of 
these  are  available  for  practical  disinfection.  At  one  time  the 
efficacy  of  chemical  disinfectants  was  probably  overrated. 
At  all  events,  in  practice  much  more  drastic  measures  than 
would  be  indicated  by  laboratory  experiments  should  be 
employed.  In  fact,  the  questions  involved  and  the  problems 
to  be  solved  in  practical  disinfection  are  often  very  different 

*For  fuller  details  on  this  subject  consult  Rosenau.  Disinfection  and  Dis- 
infectants. 1902.  Also  Gotschlich.  Disinfection.  Kolle  and  Wassermann. 
Handbuch  der  pathogenen  Mikroorganismen.  IV.  Teil.  i,  1904.  Harrington 
and  Walker.  The  Boston  Medical  and  Surgical  Journal  April  23,  1903. 
Vol.  CXLVIIL,  No.  17.  Gunther,  loc.  tit. 


248  MANUAL    OF    BACTERIOLOGY. 

from  the  destruction  of  bacteria  in  laboratory  experiments 
The  disinfection  of  a  cattle  car  or  a  Pullman  sleeper  is  a  very 
different  matter  from  the  killing  of  bacteria  on  silk  threads  or 
glass  rods  or  bits  of  paper. 

In  the  following  pages  only  those  substances  which  are  in 
common  use  or  seem  to  be  of  special  value  will  be  considered. 

Verhoeff  and  Ellis*  tested  the  following  preparations  for 
their  germicidal  power  for  S.  pyogenes  aureus :  For  one  minute 
each,  Liquor  antisepticus,  U.  S.  P.,  100  per  cent.;  Listerine,  100 
per  cent. ;  Lysol,  i  per  cent. ;  Cresylone  i  per  cent. ;  Tr.ikresol,  T\ 
per  cent. ;  Acetozone,  YQ  per  cent. ;  Alphozone,  y^  per  cent.,  and 
a  number  of  other  similar  preparations  in  different  strengths 
and  for  different  lengths  of  time.  They  found  that  while  they 
all  killed  the  bacteria,  none  were  more  efficacious  than  the 
Liquor  antisepticus.  That  any  of  the  preparations  act  as 
intestinal  germicides,  as  is  claimed  for  Alphozone  and  Acetc- 
zone,  is  improbable  from  the  fact  that  in  albuminoid  sus- 
pension the  bacteria  were  found  alive  after  twenty-four  hours' 
action,  of  these  agents. 

Mercuric  Chloride  or  Corrosive  Sublimate. — This  substance 
is  probably  more  commonly  used  than  any  other  one  germicide. 
But  Geppert  whose  work  in  this  direction  has  been  abundantly 
corroborated  by  others,  found  that  the  potency  of  corrosive 
sublimate  as  a  germicide  had  been  greatly  overrated.  The 
inhibitory  action  of  corrosive  sublimate,  on  the  other  hand,  is 
very  great,  and  the  veriest  trace  of  it  left  adhering  to  the 
bacteria  is  sufficient  to  prevent  them  from  growing.  Corro- 
sive sublimate  is  difficult  to  remove  by  ordinary  washing,  traces 
of  it  remain  even  after  very  thorough  washing.  But  if  the 
last  traces  are  removed  by  treatment  with  ammonium  suphide 
or  other  reagents  which  precipitate  the  mercury-salt  without 
themselves  injuring  the  bacteria,  growth  takes  place  even  where 
the  corrosive  sublimate  solutions  have  been  used  which  are 

*Journ.  Am.  Med.  Assn.  V.  XLVIIL,  No.  26.     1907.     pp.  2175-2176. 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          249 

apparently  efficacious.  Thus  anthrax  spores  will  not  grow 
out  in  culture  media  when  they  are  exposed  for  even  a  few 
minutes  on  silk  threads  to  the  action  of  corrosive  sublimate 
solution  of  the  strength  of  TV  per  cent,  and  then  washed  thor- 
oughly in  water  and  rinsed  in  alcohol;  but  Geppert  showed 
that  the  spores  so  treated  were  only  apparently  killed,  for  it  took 
twenty  hours,  exposure  to  corrosive  sublimate  solution  of  this 
strength  where  the  spores  were  not  dried  on  silk  threads,  but 
suspended  in  water,  and  where  the  last  trace  of  corrosive  sub- 
limate was  removed  by  treatment  with  ammonium  sulphide. 
It  is  claimed  that  its  affinity  for  albuminous  bodies  and  the 
readiness  with  which  it  combines  with  such  substances  detract 
from  its  value  for  some  purposes.  On  the  other  hand,  many 
observers  claim  that  the  albuminous  combinations  formed 
under  such  circumstances  are  soluble  in  an  excess  of  albuminous 
.fluid,  and  that  its  value  as  a  germicide  is  not  affected  thereby. 
To  obviate  this  possible  difficulty  it  is  customary  in  practice 
to  combine  the  bichloride  of  mercury  with  some  substance 
that  will  prevent  the  precipitation  of  the  mercury-salt  by  al- 
bumin. For  this  purpose  5  parts  of  any  one  of  the  following  sub- 
stances to  i  part  of  bichloride  of  mercury  may  be  used — hydro- 
chloric acid,  tartaric  acid,  sodium  chloride,  potassium  chloride, 
or  ammonium  chloride.  A  very  practical  stock-solution  for 
laboratory  purposes  has  the  following  composition: 

Hydrochloric  acid 100  c.c. 

Bichloride  of  mercury 20  grams. 

Five  c.c.  in  a  liter  of  water  makes  a  solution  of  about  i-iooo  strength. 

Mercuric  Iodide. — An  extremely  high  antiseptic  value  has 
been  placed  on  this  substance  by  Miquel,  who  claims  that  the 
most  resistant  spores  are  prevented  from  developing  in  a  cul- 
ture-medium containing  1-40,000.  In  combination,  as  potas- 
sio-mercuric  iodide,  it  has  been  used  in  soaps  (McClintock) 
with  very  favorable  results.  The  substance  is  not  extensively 


2CJO  MANUAL    OF    BACTERIOLOGY. 

employed,  and  further  investigation  is  necessary  to  determine 
its  true  value. 

Attempts  are  being  made  to  manufacture  combinations  of 
mercury  and  other  powerful  metallic  germicides  with  organic 
acid  and  basic  bodies,  the  purpose  being  to  utilize  the  metallic 
base  in  greater  strength  without  injury  to  the  living  tissues. 
Such  compounds  are  exemplified  by  mercurol,  said  to  be  a 
combination  of  mercury  with  nucleinic  acid,  and  to  possess 
active  germicidal  properties,  great  penetrating  power,  and  no 
injurious  effect  on  living  tissue.  It  is  also  said  to  have  a  par- 
ticularly destructive  action  upon  the  gonococcus. 

Silver  Nitrate. — This  salt  probably  occupies  the  next  posi- 
tion to  the  bichloride  of  mercury  in  germicidal  power.  Behr- 
ing  claims  it  to  be  superior  to  bichloride  of  mercury  in  al- 
buminous fluids.  The  anthrax  bacillus  is  killed  by  a  solution 
of  1-20,000  after  two  hours'  exposure.  At  least  forty-eight 
hours'  exposure  to  a  1-10,000  solution  is  required  to  kill  the 
spores  of  anthrax.  It  is  very  irritating,  and  possesses  strong 
affinities  for  chlorides,  forming  with  them  insoluble  chloride  of 
silver,  a  salt  without  germicidal  value.  For  these  reasons  the 
use  of  silver  nitrate  is  limited.  In  the  solutions  usually  em- 
ployed for  douching  the  cavities  of  the  body  the  available 
silver  nitrate  is  immediately  converted  into  the  insoluble  chlo- 
ride, and  little  if  any  germicidal  action  takes  place.  To  this 
fact  may  be  ascribed  the  varying  clinical  results  reported. 

Many  semi-proprietary  silver  compounds  are  on  the  market, 
introduced  to  replace  the  nitrate  and  its  objectionable  features. 
The  most  important  are  argentamin,  argonin,  protargol  and 
argyrol,  all  organic  silver  combinations.  They  do  not  combine 
with  chlorides,  are  less  irritating  than  the  nitrate  and,  not  coagu- 
lating albumin,  they  possess  greater  penetrating  power. 
Clinical  reports  and  investigations  have  been  so  contradictory 
thus  far  that  their  value  cannot  be  readily  estimated. 

Carbolic  Acid. — One  of  the  most  important  and  most  widely 


DISINFECTION,    STERILIZATION   AND    ANTISEPSIS.          251 

used  germicides.  It  is  usually  employed  in  strengths  of  from 
i  to  5  per  cent.  A  3  per  cent,  solution  will  sometimes  kill  the 
spores  of  anthrax  after  two  days'  exposure  (see  Bacillus  anthra- 
cis,  Part  IV.) .  In  the  absence  of  spores  the  anthrax  bacillus 
is  destroyed  by  a  i  per  cent,  solution  in  one  hour.  The  less 
resistant  pus  cocci  are  destroyed  rapidly  by  a  2  per  cent,  solu- 
tion. Combination  with  an  equal  proportion  of  hydrochloric 
acid  enhances  the  efficacy  of  carbolic  acid  to  a  marked  extent. 
This  is  due  to  the  prevention  of  albuminous  combinations, 
thus  allowing  greater  penetration  of  the  germicide. 

McBryde*  found  that  the  admixture  of  lime  to  carbolic  acid 
lessened  the  germicidal  potency  of  the  latter.  He  furthermore 
found  that  liquor  cresolis  comp.,  U.  S.  P.,  a  liquid  soap  con- 
taining 50  per  cent,  cresol,  misciblein  all  proportions  with  water, 
is  a  more  potent  germicide  than  carbolic  acid.  Liquor  cres. 
comp.  made  from  cresol  of  high  boiling-points  is  more  potent 
than  that  made  of  cresol  of  lower  boiling-points.  That  made 
with  cresol  of  187  to  189°  C.  is  nearly  one  and  one-half  times 
greater  than  carbolic  acid. 

Many  other  substances  closely  related  to  carbolic  acid  are 
used  and  possess  marked  germicidal  properties.  Among  them 
may  be  mentioned  creolin,  cresol  and  lysol.  They  are  all 
slightly  superior  to  carbolic  acid  in  actual  germicidal  value. 

Aniline  Dyes. — Many  of  these  substances,  notably  pyok- 
tanin  (methyl- violet),  possess  germicidal  properties.  Mala- 
chite green  is  said  to  possess  even  greater  germicidal  value  than 
pyoktanin.  Methylene-blue  also  possesses  considerable  germi- 
cidal power. 

Formalin  is  a  40  per  cent,  aqueous  solution  of  formaldehyde. 
Results  of  the  earlier  investigations  seemed  to  show  that  for- 
maldehyde possessed  remarkably  high  germicidal  properties, 
but  1  ater  experiments  have  failed  to  corroborate  these.  In  solu- 
tions  of  i-iooo  an  exposure  of  twenty-four  hours  is  necessary 

*Dept.  Agr.,  Bu.  An.  In.,  Bui.   100.     May  31.  1907. 


252  MANUAL   OF    BACTERIOLOGY. 

to  destroy  the  Staphylococcus  pyogenes  aureus,  while  1-5000 
is  sufficient  to  restrain  its  growth  (Slater  and  Rideal) .  Its  use 
in  a  gaseous  form  as  a  house-disinfectant  is  by  far  the  most  im- 
portant application  at  the  present  time. 

In  vaporizing  the  gas  many  methods  have  been  employed. 
Simple  evaporization  of  solutions  without  heat  cannot  be 
relied  upon,  for  the  solid,  polymerized  paraformaldehyde  is 
easily  formed  under  these  circumstances.  Better  results  can 
be  obtained  with  the  aid  of  heat,  although  polymerization  is  apt 
to  occur  unless  evaporation  is  rapid.  To  produce  the  best 
results  it  has  been  found  necessary  to  use  special  forms  of 
lamps  or  generators  for  its  production,  a  few  of  which  are 
mentioned  below. 

Gotschlich*  has  carefully  summed  up  the  results  obtained 
with  formalin  and  its  various  preparations.  He  finds  that 
while  authorities  agree  in  attributing  very  powerful  inhibitory 
action  to  formaldehyde,  they  fail  to  get  very  powerful  germi- 
cidal  action.  Even  -^-Q^-Q-Q  shows  marked  inhibition,  and  y-0-J-jnr 
complete  inhibition  of  bacterial  growth  in  albuminous 
media  even,  varying  of  course  with  different  bacteria.  But  in 
contrast  to  this  2.5  to  5  per  cent,  was  required  to  kill  the  same 
organisms.  The  action  of  formalin  is  greatly  increased  by  heat- 
ing. Anthrax  spores  were  killed  in  one  and  one-half  hours  in 
12.5  to  15  per  cent,  formalin  at  room  temperature,  in  the  same 
solution  they  were  killed  in  thirty  minutes  at  35°  C.,  and  in  five 
minutes  at  52°  C.  Lysoform,  a  perfumed  soap  containing 
formalin  was  found  to  be  weak;  septoform,  a  similar  soap 
preparation  was  found  to  be  somewhat  m  >re  powerful ;  it  has 
the  remarkable  property  of  being  more  strongly  germicid?!  for 
anthrax  spores  than  for  the  pus  cocci.  Other  proprietary 
preparations  were  also  found  to  be  weak  in  germicidal  power, 
others  again  more  powerful  Acrolein  which  is  allylaldehyde 
is  said  to  have  greater  germicidal  power  than  formalin. 

*Kolle  and  Wassermann.     Loc.  cit 


DISINFECTION,    STERILIZATION    AND   ANTISEPSIS.          253 

A  great  many  methods  have  been  devised  for  using  formalin 
for  room  disinfection  in  the  form  of  vapor.  These  vary  all 
the  way  from  simply  hanging  up  sheets  wet  with  formalin  to 
more  or  less  elaborate  lamps  for  burning  methyl  alcohol  and 
converting  it  into  formaldehyde.  The  two  most  important  con- 
ditions to  fulfill  in  the  use  of  formaldehyde  as  a  gas  are  tempera- 
ture and  moisture.  The  room  to  be  disinfected  should  be 
warmed  and  the  air  saturated  with  moisture. 

Hill*  finds  an  explanation  of  the  discrepancy  in  results 
obtained  by  different  observers  in  the  condition  of  the  bacteria 
as  to  dryness  or  moisture,  and  concludes  that  for  practical  dis- 
infection the  destruction  of  bacteria  thrown  off  from  the  patient 
in  any  way  for  some  time  before  the  disinfection  of  the  premises 
should  be  aimed  at. 

Sanitary  Construction  Company's  Lamp. — This  lamp  consists 
of  a  tank  to  hold  the  formaldehyde  solution,  and  a  spiral  tube 
by  which  the  solution  is  slowly  conducted  through  a  flame  and 
vaporized.  The  necessary  amount  of  solution  is  placed  in  the 
tank  and  the  apparatus  started,  outside  the  room,  the  gas  being 
conducted  through  the  keyhole  by  a  suitable  tube. 

Schering  Lamp. — In  this  form  of  lamp  formaldehyde  is 
generated  by  the  decomposition  of  paraform  or  paraformalde- 
hyde,  a  polymeric  modification  of  formaldehyde,  occurring  as 
a  white  salt.  It  is  decomposed  by  heat,  giving  off  formaldehyde 
gas.  It  is  placed  on  the  market  in  the  form  of  tablets,  each 
one  of  which  yields  a  definite  amount  of  gas.  The  lamp  con- 
sists of  a  small  iron  tray  for  the  reception  of  tablets,  and  so 
arranged  above  the  heating  apparatus  that  sufficient  draught 

*Journ.  Infectious  Diseases.     Supplement  2.     Feb.,  1906.,  p.  210. 

Other  references  to  formaldehyde  in  disinfection  are: 

Ravenel.  Report  of  the  American  Public  Health  Association.  Vol.  XXVIII. , 
p.  221. 

Hill,  Eben.     Vol.  XXIX.,  p.  208. 

Rosenau.  Disinfection  and  Disinfectants.  Pub.  Health  and  Mar.  Hosp. 
Ser. 

Evans.     Journ.  Infec.  Diseases.     Sup.  No.  3,  May,  1907. 

Herzog.     Centralblatt  f.  Bakteriologie ,  etc.     Orig.  XXXIV.,  2,   1903. 


254 


MANUAL    OF    BACTERIOLOGY. 


is  created  to  carry  off  the  gas  as  rapidly  as  formed.  In  opera- 
ting, a  sufficient  number  of  tablets  are  placed  on  the  tray,  the 
lamp  lighted  and  placed  in  the  room  to  be  disinfected. 

Methyl- alcohol  Lamps. — Several  of  these  lamps  are  on  the 
market,  all  operating  on  the  well-known  principle  of  the  oxida- 
tion of  wood-alcohol  to  formaldehyde  when  the  alcohol  is 
vaporized  by  projection  against  a  heated,  platinized,  asbestos 
disk.  In  operating  such  an  apparatus,  the  alcohol  is  lighted 
until  the  asbestos  disk  becomes  hot.  The  flame  is  then  extin- 
guished; the  heat  from  the  disk  is  sufficient  to  vaporize  the 
alcohol,  which  undergoes  oxidation  and  keeps  the  disk  at  a 
red  heat.  When  the  apparatus  is  operating  in  a  satisfactory 
manner,  the  room  is  closed  and  disinfection  allowed  to  proceed. 
It  must  be  said,  however,  that  it  is  difficult  to  estimate  or 
control  the  amount  of  formaldehyde  evolved  in  generators  of 
this  type. 

Formaldehyde  Candles. — Mixtures  of  paraformalhyde  and 
paraffin  or  other  combustibles,  which  may  be  moulded  into 
candles,  each  enclosed  in  a  tin  case,  make  a  convenient  ap- 
paratus to  generate  formaldehyde  gas  for  room  disinfection. 
The  candle  is  placed  in  a  suitable  fireproof  dish,  it  is  then  ig- 
nited, and  generation  of  the  gas  is  allowed  to  proceed  in  the 
tightly  closed  room. 

Sulphur  Dioxide. — This  substance  is  used  extensively  for 
house  disinfection,  and  is  usually  prepared  by  burning  sulphur. 
Much  difference  of  opinion  exists  regarding  the  value  of  it  as  a 
disinfectant.  The  spores  of  anthrax  are  not  killed  by  several 
days'  exposure  to  the  liquefied  gas.  Anthrax  and  other 
bacilli  are  destroyed  in  thirty  minutes  when  exposed  on  moist 
threads  in  an  atmosphere  containing  one  volume  per  centum  of 
the  gas.  An  exposure  of  twenty-four  hours  in  an  atmosphere 
containing  four  volumes  per  centum  of  the  gas  will  destroy  the 
organisms  of  typhoid  fever,  diphtheria,  cholera  and  tubercu- 
losis. The  presence  of  moisture  greatly  enhances  the  activity 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          255 

of  the  disinfectant,  owing  to  the  formation  of  the  more  ener- 
getic sulphurous  acid. 

For  the  destruction  of  insects,  such  as  mosquitoes,  this  agent 
is  superior  to  formaldehyde.  Its  application  for  this  purpose 
is  important  in  preventing  the  spread  of  yellow  fever  and 
malaria. 

In  practice,  at  least  three  pounds  of  sulphur  per  1000  cubic 
feet  should  be  used,  and  moisture  must  be  present.  This  latter 
requirement  can  be  fulfilled  by  evaporating  several  quarts  of 
water  within  the  tightly  closed  room  just  prior  to  generating 
the  gas.  In  using  powdered  or  flowers  of  sulphur,  the  neces- 
sary amount  is  placed  on  a  bed  of  sand  or  ashes  in  an  iron  pot, 
which  should  be  supported  on  some  bricks  in  a  pan  or  other 
vessel  containing  an  inch  or  two  of  water.  The  sulphur  is 
ignited  by  means  of  some  glowing  coals,  or  by  moistening  with 
alcohol  and  applying  a  match.  Difficulty  is  often  experienced 
in  keeping  the  sulphur  burning,  and  for  this  reason  it  is  surer 
and  more  convenient  to  use  the  so-called  sulphur  candles  now 
on  the  market.  In  operating  with  these,  a  sufficient  number 
are  placed  on  bricks  in  a  pan  of  water  and  the  wicks  lighted. 
Liquefied  sulphur  dioxide  may  be  used,  and  can  now  be  ob- 
tained in  convenient  tin  receptacles  containing  a  sufficient 
quantity  for  the  disinfection  of  an  ordinary  room.  The  can  is 
opened  by  cutting  through  a  soft  metal  tube  projecting  from 
the  top.  The  fluid  vaporizes  at  the  room  temperature,  and  it 
is  simply  necessary  to  place  the  pan  in  a  convenient  porcelain 
dish  and  allow  the  fluid  to  evaporate. 

Sulphur  dioxide  is  objectionable  on  account  of  its  lack  of 
power  when  dry,  and  on  account  of  its  corrosive  action  on 
metal  and  its  bleaching  effect  on  hangings  and  draperies  in 
the  presence  of  moisture;  it  is,  therefore,  preferable  to  use 
formaldehyde  when  possible. 

Chlorine. — A  very  active  gaseous  disinfectant,  particularly 
in  the  presence  of  moisture.  An  atmosphere  containing  i  per 


256  MANUAL    OF    BACTERIOLOGY. 

cent,  of  the  dry  gas  is  fatal  to  anthrax  spores  in  three  hours. 
The  anthrax  bacillus  is  killed  in  twenty-four  hours  by  ex- 
posure to  a  moist  atmosphere  containing  the  gas  in  the  pro- 
portion of  1-2500.  The  bacillus  of  tuberculosis  is  killed  by  an 
exposure  of  one  hour  to  a  moist  atmosphere  containing  the  gas 
in  the  proportion  of  1-200.  Extremely  minute  quantities  in 
solution  will  prevent  the  development  of  putrefactive  organ- 
isms. The  substance  has  been  used  for  house  and  ship  disin- 
fection, but  is  now  seldom  employed  on  account  of  its  extremely 
irritating  properties  and  the  difficulty  of  handling. 

Bromine. — Used  in  the  gaseous  and  liquid  form.  The  dry 
vapor  possesses  but  little  disinfectant  power;  when  moist  it 
is  much  more  efficient.  In  saturated  acqueous  solution  it  will 
kill  the  anthrax  bacillus  in  twenty-four  hours. 

Calcium  Hypochlorite,  usually  known  as  Chloride  oj  Lime. — 
This  is  a  most  practical  and  valuable  disinfectant,  depending 
for  its  efficiency  on  the  available  chlorine  contained  in  it.  Its 
alkalinity  favors  penetration,  and  for  many  purposes  it  can- 
not be  excelled.  A  i  per  cent,  solution  will  destroy  anthrax 
spores  in  one  hour.  A  solution  of  the  same  strength  will  disin- 
fect typhoid  stools  in  ten  minutes. 

Lime. — The  addition  of  o.i  per  cent,  of  unslaked  lime  to 
fluid-cultures  of  the  typhoid  bacillus  and  cholera  spirillum  will 
render  them  sterile  in  four  or  five  hours.  Typhoid  dejecta  are 
sterilized  in  six  hours  by  the  addition  of  3  per  cent,  of  slaked 
lime;  the  addition  of  6  per  cent,  will  accomplish  the  same  result 
in  two  hours.  A  convenient  form  for  practical  use  is  an  aqueous 
mixture  containing  20  per  cent,  of  lime — so-called  milk  of 
lime.  Typhoid  and  cholera  dejecta  are  sterilized  in  one  hour 
after  the  addition  of  20  per  cent,  of  this  mixture.  In  practice 
it  is  safer  to  use  a  considerable  excess  of  lime.  From  the  fore- 
going facts  it  would  seem  probable  that  lime  or  whitewash  as 
ordinarily  applied  would  possess  disinfectant  properties.  Ex- 
perimental work  has  demonstrated  this  to  be  a  fact.  The 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          257 

organisms  of  anthrax,  glanders  and  the  pus  cocci  were  de- 
stroyed within  twenty-four  hours  by  one  application.  For 
spore-forming  organisms  and  the  bacillus  of  tuberculosis  the 
power  is  not  so  great,  the  latter  organism  not  being  destroyed 
by  three  applications  of  the  whitewash.  This  is  due,  perhaps, 
to  the  large  amount  of  fatty  matter  in  the  bacillus  of  tubercu- 
losis, and  suggests  the  possibility  of  enhancing  the  efficacy  of 
the  lime  by  the  addition  of  a  small  proportion  of  caustic  alkali. 

Hydrogen  Peroxide.  — This  substance  is  placed  on  the  mar- 
ket in  solutions  varying  in  strength  from  10  to  30  volumes; 
the  mode  of  expression  indicating  that  corresponding  solutions 
will  liberate  ten  to  thirty  times  their  volume  of  oxygen  when 
appropriately  treated.  •  It  possesses  the  property  of  rapidly 
oxidizing  purulent  secretions,  and  on  this  account  is  much  used 
for  cleansing  infected  wounds.  It  deteriorates  in  strength  so 
rapidly  that  only  fresh  solutions  of  known  strength  should  be 
used. 

Potassium  Permanganate. — Koch  asserts  that  a  3  per  cent, 
solution  will  destroy  anthrax  spores  in  twenty-four  hours,  but 
that  a  i  per  cent,  solution  cannot  be  depended  upon  to  kill 
pathogenic  organisms.  Its  disinfectant  value  in  practice  is 
very  low  on  account  of  its  ready  decomposition  by  inert  ma- 
terial. In  the  dilute  solutions  usually  used  for  medicinal  in- 
jections and  irrigations  no  disinfectant  action  occurs. 

lodojorm. — This  substance  possesses  little  if  any  disinfectant 
power.  It  is  mildly  antiseptic  in  moist  wounds,  due  to  the 
gradual  liberation  of  small  quantities  of  iodine. 

Boric  Acid. — This  material  possesses  practically  no  disin- 
fectan  t  power.  It  is  a  mild  antiseptic  when  applied  as  an 
undiluted  powder  to  wounds.  A  saturated  aqueous  solution 
is  much  used,  and  is  weakly  antiseptic. 

Essential  Oils. — Many  of  these  bodies  possess  germicidal 
value,  notably  the  oils  of  cinnamon  and  cloves.  The  oil  of 
mustard  is  also  a  valuable  disinfectant,  but  so  irritating  that 


258  MANUAL    OF    BACTERIOLOGY. 

the  pure  oil  cannot  be  used.  The  use  of  powdered  mustard, 
in  the  autopsy-room  will  remove  the  foul  odor  from  the  hands 
more  rapidly  and  completely  than  any  other  means. 

Coal  Oil  or  Petroleum. — While  the  disinfectant  value  of  this 
substance  is  slight,  its  use  in  destroying  the  larvae  of  insects, 
such  as  the  mosquito,  has  given  it  an  important  position  in 
preventing  the  spread  of  malaria  and  yellow  fever.  A  small 
amount  poured  on  a  stagnant  pool  rapidly  spreads  over  the 
surface  and  effectually  destroys  such  larvae. 

Ferrous  Sulphate  (Copperas). — This  salt  has  been  much  used, 
but  possesses  only  feeble  disinfectant  powers.  A  3  per  cent, 
solution  requires  three  days  to  kill  the  bacillus  of  typhoid  fever. 
On  account  of  its  affinity  for  ammonia  and  sulphides  it  is  an 
efficient  deodorizer  for  temporary  use,  but  cannot  be  relied 
upon  to  kill  the  bacteria  producing  the  noxious  gases. 

Cupric  Sulphate  (Blue  Vitriol}. — This  salt  is  quite  an  effi- 
cient disinfectant.  In  a  solution  of  1-3000  the  spirillum  of 
cholera  is  destroyed  in  ten  minutes.  A  5  per  cent,  solution  will 
kill  the  typhoid  bacillus  in  ten  minutes.  A  solution  of  from  2 
to  3  per  cent,  in  strength  can  be  relied  upon  to  destroy  all 
pathogenic  organisms  that  do  not  form  spores. 

Zinc  Sulphate. — This  salt  is  a  very  feeble  disinfectant.  Pus 
cocci  are  not  destroyed  in  two  hours  by  a  20  per  cent,  solution. 
As  a  deodorizer  it  has  about  the  same  value  and  acts  in  the 
same  way  as  ferrous  sulphate. 

Zinc  Chloride. — A  2  per  cent,  solution  will  kill  pus  cocci 
after  an  exposure  of  two  hours.  It  is  therefore  a  much  more 
powerful  disinfectant  than  the  sulphate. 

Disinfection  of  the  Mouth. — Wadsworth*  found  that  for 
mouth  disinfection  in  pneumonia,  of  all  the  antiseptics  in  com- 
mon use  alcohol  alone  proved  efficient  when  tested  on  the  pneu- 
mococcus  He  tested  a  long  list  of  various  preparations,  but 
these  were  all  counteracted  by  the  mucus  in  the  mouth. 

*Journ.  Infectious  Diseases.     Vol.   III.,   p.   774. 


DISINFECTION,    STERILIZATION    AND    ANTISEPSIS.          259 

Disinfection  of  Dejecta  and  Urine. — A  4  per  cent,  solution 
of  calcic  hypochlorite  (chloride  of  lime)  is  most  efficient  and 
rapid  for  this  purpose.  A  convenient  solution  contains  six 
ounces  of  the  salt  to  one  gallon  of  water.  The  excreta  should 
be  received  in  a  suitable  vessel  and  immediately  mixed  with  an 
equal  bulk  of  the  disinfectant.  The  contents  of  the  vessel 
should  be  allowed  to  stand  for  one  hour  before  emptying. 
A  20  per  cent,  milk  of  lime  is  just  as  efficient,  and  possesses 
the  advantage  of  cleanliness  and  lack  of  odor.  It  should  be 
used  in  the  same  quantity  and  allowed  to  act  for  the  same 
length  of  time.  A  5  per  cent,  solution  of  carbolic  acid  may 
be  used,  but  should  be  allowed  to  act  for  at  least  four  hours. 

Disinfection  of  Sputum. — The  chemical  disinfection  of  tuber- 
culous sputum  is  somewhat  difficult  on  account  of  the  large 
amount  of  albumin  in  it  and  the  fatty  matter  associated  with 
the  bacillus  of  tuberculosis.  Dilute  solutions  of  bichloride  of 
mercury  are  apt  to  be  decomposed  and  rendered  inert  by  the 
albumin.  Carbolic  acid  is  open  to  the  same  objection,  but  its 
combination  with  hydrochloric  acid  can  be  used  successfully 
in  a  strength  of  5  per  cent.  each.  Milk  of  lime  cannot  be  relied 
upon  for  this  purpose.  A  4  per  cent,  solution  of  calcic  hypo- 
chlorite (chloride  of  lime)  is  the  best  for  general  use,  and  the 
spit-cup  should  be  kept  nearly  full  of  this  solution.  Sputum 
may  also  be  disinfected  by  exposure  to  the  action  of  steam  in 
the  sterilizer  or  by  boiling  for  fifteen  minutes.  If  napkins  or 
old  pieces  of  cloth  are  used  for  the  reception  of  sputum,  they 
may  be  immediately  destroyed  in  a  fire. 

Disinfection  after  Postmortems. — After  autopsies  on  infec- 
tious cases  it  is  necessary  to  disinfect  the  table  and  fluid  prod- 
ucts coming  from  it  prior  to  emptying  into  the  sewer.  The 
table  may  be  disinfected  by  a  liberal  sprinkling  with  4  per  cent, 
calcic  hypochlorite  solution.  All  fluids  should  be  treated  with 
an  equal  quantity  of  the  same  solution.  The  table  should  not 
be  cleaned  for  at  least  one  hour  after  application  of  the  disin- 


260  MANUAL    OF    BACTERIOLOGY. 

fectant.  The  same  rule  applies  to  the  disinfection  of  the 
fluids — an  exposure  of  at  least  one  hour  to  the  disinfectant 
before  final  disposition. 

The  Cadaver  in  Contagious  Diseases. — In  cases  of  death 
from  a  contagious  disease  all  the  orifices  of  the  body  should  be 
packed  with  cotton  soaked  in  a  strong  solution  (1-500)  of 
bichloride  of  mercury,  the  skin  washed  with  a  i-iooo  solution, 
and  the  cadaver  wrapped  in  a  sheet  wet  with  the  same.  The 
funeral  should  be  private  and  the  body  disposed  of  within 
twenty-four  hours,  preferably  by  cremation. 

House  Disinfection. — After  infectious  disease  it  is  essential 
that  the  house  or  the  apartment  in  which  the  patient  has  been 
confined  should  be  disinfected.  '  It  is  rarely  necessary  to  carry 
out  the  process  in  more  than  two  rooms;  but  should  it  be  so, 
the  process  can  be  applied  to  the  whole  house. 

After  thorough  bathing  of  the  patient,  preferably  with  an 
antiseptic  soap,  the  individual  should  be  wrapped  in  a  clean 
sheet  and  removed  to  a  clean  room.  All  articles  or  materials 
that  are  of  little  value  should  be  destroyed.  All  bedding, 
towels  and  the  like  should  be  placed  in  wooden  tubs  and 
covered  with  a  i-iooo  solution  of  bichloride  of  mercury.  The 
room  should  then  be  made  as  nearly  airtight  as  possible;  this 
can  be  accomplished  by  pasting  strips  of  paper  over  registers, 
cracks,  spaces 'between  window-sashes  and  the  like.  Formal- 
dehyde gas  is  then  passed  through  the  keyhole  into  the  room 
(or  it  may  be  generated  by  formaldehyde  candles)  in  sufficient 
quantity  to  destroy  the  infectious  element. .  The  room  should 
be  sealed  for  at  least  twelve  hours,  after  which  time  it  may  be 
opened  and  aired.  The  process  is  completed  by  washing  all 
exposed  surfaces  in  the  room  with  i-iooo  bichloride  of  mer- 
cury. This  latter  requirement  is  not  essential  if  the  gaseous 
disinfection  has  been  complete,  but  since  we  have  no  absolute 
knowledge  on  this  point,  the  secondary  washing  should  be 
carried  out.  This  method  can  be  considered  reliable  for  sur- 


DISINFECTION,    STERILIZATION   AND    ANTISEPSIS.          261 

face  disinfection,  but  for  the  interior  of  mattresses  and  stuffed 
furniture-cushions  it  is  not  certain.  In  all  cases  where  ab- 
solute disinfection  is  demanded,  such  articles  must  be  ripped 
apart  and  loosely  exposed  to  the  gas  or  sterilized  by  steam 
under  pressure.  Instead  of  formaldehyde,  sulphur  dioxide 
may  be  used  for  room  disinfection,  but  in  the  light  of  present 
knowledge  the  formaldehyde  method  is  superior. 

Stokes  and  Stubbs*  found  that  reinfection  of  premises  which 
had  been  disinfected  with  formalin  occurred  in  the  2807  cases 
of  diphtheria  which  were  made  the  subject  of  inquiry  in  2.35 
per  cent,  in  the  same  year:  in  the  2739  cases  of  scarlet  fever 
similarly  examined  reinfection  of  the  premises  occurred  in 
2.55  per  cent. 

*W.  R.  Stokes  and  W.  P.  Stubbs.     Maryland  Medical  Journal.     Feb.,   1907. 


CHAPTER  VIII. 
SURGICAL  ANTISEPSIS. 

MANY  important  advances  in  surgery  have  been  made  pos- 
sible by  the  practical  application  of  the  principles  of  steriliza- 
tion and  antisepsis  set  forth  in  the  preceding  chapter.  Pre- 
vious to  the  introduction  of  antisepsis  in  surgery  there  was  im- 
minent risk  of  suppuration  and  septicemia  even  in  the  most 
insignificant  operations,  and  many  of  the  capital  operations 
which  are  now  daily  performed  without  serious  risks  were  at 
that  time  so  often  attended  by  fatal  consequences  due  to  in- 
fection that  the  patient  and  the  surgeon  were  deterred  from 
taking  the  chances. 

The  four  sources  of  infection  in  surgery  are  from  the  bacteria 
in  the  air  of  the  room  in  which  the  operation  is  performed,  the 
bacteria  on  the  surface  of  the  patient's  body,  the  bacteria  on 
the  hands  of  the  surgeon,  and  finally  the  bacteria  on  the  in- 
struments with  which  the  operation  is  performed  and  on  the 
suture  material  and  dressings.  All  of  these  sources  of  infec- 
tion have  to  be  taken  into  account  and  guarded  against. 

In  the  modern  operating-room  the  danger  from  infection 
through  the  air  is  reduced  very  greatly.  The  room  is  so  con- 
structed and  fitted  with  tables  and  other  necessary  furniture 
that  everything  may  be  washed  and  rubbed  with  antiseptic 
solutions.  Everything  is  done  to  avoid  the  accumulation  of 
dust  and  to  prevent  the  distribution  of  dust  in  the  air.  The 
walls  and  floor  are  wiped  with  moist  cloths  since  the  bacteria 
cannot  be  blown  from  moist  surfaces  by  any  ordinary  circula- 
tion of  the  air  nor  by  even  any  draught  that  is  ordinarily  en- 
countered in  an  operating  room.  On  the  contrary  the  bacteria 

262 


SURGICAL    ANTISEPSIS.  263 

become  very  easily  disseminated  through  the  air  where  every- 
thing is  dry.  Even  walking  through  the  room  is  sufficient  to 
stir  up  the  bacteria  from  a  dry,  dusty  floor.  The  air  is,  how- 
ever, probably  not  as  serious  a  source  of  danger  as  the  others 
mentioned,  for  there  are  not  as  many  bacteria,  specially  not 
as  many  pathogenic  bacteria  found  in  the  air  as  was  at  one 
time  supposed  to  be  present  there.  In  the  early  days  of  anti- 
septic surgery  the  air  was  supposed  to  be  such  a  dangerous 
source  of  infection  that  operations  were  performed  under  a 
carbolic  acid  spray.  The  seat  of  operation  and  the  hands  of 
the  operator  were  sprayed  continuously  throughout  the  opera- 
tion. This  practice  is  no  longer  employed.  Still  the  danger 
from  the  air  infection  although  less  serious  than  was  at  one 
time  supposed  should  nervertheless  not  be  overlooked. 

The  danger  from  the  introduction  of  bacteria  into  a  surgical 
wound  from  the  surface  of  the  patient's  own  body  must  be  care- 
fully guarded  against.  There  are  apt  to  be  pus-producing 
cocci  on  the  surface  as  well  as  deeper  in  the  layers  of  the  epider- 
mis. The  staphylococcus  epidermidis  albus  (Welch)  is  found 
to  be  quite  constantly  present  in  the  deeper  layers  of  the  epider- 
mis, and  is  apt  to  give  rise  to  the  stich  abscesses  which  so  often 
form  around  sutures  in  even  the  most  successful  operations. 
Surgical  cases  also  are  common  in  which  bacteria  have  been  in- 
troduced in  an  accidental  wound  either  at  the  time  of  the  ac- 
cident, as  where  the  patient  has  received  an  accidental  wound 
with  some  infected  instrument,  or  where  the  patient  has  been 
thrown  to  the  ground  and  had  dirt  rubbed  into  the  wound,  or 
where  the  patient  has  been  wounded  with  a  dirty  splinter  of 
wood  or  dirty  nail  or  the  infection  of  the  wound  may  take  place 
later  by  the  dressing  of  the  wound  with  dirty  rags.  In  every 
case  the  seat  of  operation  should  be  disinfected  as  thoroughly 
as  possible.  Unfortunately  this  is  a  difficult,  not  say  an  impos- 
sible task  in  some  case  at  least.  Certain  parts  of  the  body  are 
much  more  easily  disinfected  than  other  parts.  The  abdomen 


264  MANUAL    OF    BACTERIOLOGY. 

is  comparatively  easily  disinfected,  but  the  epidremis  of  the 
hands  is  very  difficult.  The  mucous  membranes  are  dif- 
ficult to  disinfect  for  the  reason  of  there  delicate  structure,  the 
interference  of  the  secretions  with  the  action  of  disinfectants 
which  precipitate  them,  and  moreover  because  the  disinfectants 
have  so  little  power  of  penetration  below  the  surface.  The 
lack  of  penetration  also  interferes  with  the  action  of  disinfec- 
tants on  the  epidermis.  The  application  of  disinfectants  to  the 
surface  does  little  good  unless  it  is  preceded  by  thorough  scrub- 
bing with  soap  and  water.  The  mechanical  washing  is  of  more 
importance  than  the  application  of  disinfectants  in  any  case. 
In  fact  some  have  advised  the  use  of  scrubbing  with  soap  and 
water  alone,  but  it  would  seem  rational  to  suppose  that  the  ad- 
ditional use  of  disinfectants  would  be  of  advantage. 

It  follows  from  what  has  been  said  that  it  is  very  difficult  for 
the  operator  to  sterilize  his  hands,  or  at  least  to  be  sure  that  the 
hands  are  sterile.  Many  different  methods  have  been  recom- 
mended for  the  purpose,  all  of  them  having  this  in  common 
that  they  recognize  the  importance  of  thorough  scrubbing 
with  soap  and  water,  with  particular  attention  to  the  folds  of 
the  epidermis  around  the  nails  and  under  the  nails.  All  seem 
to  agree  that  the  subsequent  treatment  of  the  hands  with 
alcohol  as  advised  by  Furbringer,  no  matter  what  subsequent 
treatment  is  employed,  is  very  efficacious.  The  strength  of 
alcohol  recommended  is  at  least  80  per  cent.,  and  the  hands 
should  be  washed  in  the  alcohol  for  a  few  minutes.  After  the 
scouring  with  soap  and  water  and  washing  in  alcohol,  some 
advise  no  further  sterilization,  others  prefer  to  use  various  an- 
tiseptics. The  alcohol  has  not  only  germicidal  value,  but  it 
also  frees  the  hands  from  grease  and  thus  prepares  the  skin 
for  treatment  with  antiseptics.  But  it  has  been  pointed  out 
that  the  adjuvant  action  of  alcohol  when  used  along  with  other 
antiseptics  cannot  be  attributed  to  its  solvent  action  on  the  fat 
alone,  for  ether  is  a  better  solvent  for  fat  than  alcohol  is,  and 


SURGICAL   ANTISEPSIS.  265 

yet  ether  has  been  found  not  to  aid  the  action  of  antiseptics  on 
the  hands  to  the  same  extent  as  alcohol.  What  has  just  been 
stated  in  regard  to  the  sterilization  of  the  hands  of  the  surgeon 
applies,  of  course,  also  to  the  field  of  operation. 

The  use  of  rubber  gloves  and  of  various  other  impervious 
coatings  for  the  hands  are  extensively  employed,  and  the  rub- 
ber gloves  give  excellent  results  where  it  is  feasible  to  use  them. 

The  various  coatings  which  have  been  recommended  from 
time  to  time  seem  not  to  have  found  general  adoption  for  the 
reason  that  all  of  them  soon  crack  or  wear  off  during  the  opera- 
tion. The  use  of  sterilized  rubber  gloves  has  the  double  ad- 
vantage of  lessening  the  chances  of  infecting  the  patient  and  of 
insuring  the  operator  against  infecting  himself  when  handling 
wounds  which  are  already  infected,  as  in  the  case  of  syphilitic 
infection.  Rubber  gloves  may  be  sterilized  by  steam  or  by  the 
use  of  disinfecting  solutions.  Ordinary  cotton  gloves  seem 
also  to  afford  a  considerable  degree  of  protection  for  the 
patient  and  the  operator. 

Materials  used  for  sutures  and  dressings  are  best  sterilized 
by  steam,  preferably  in  an  autoclave.  All  sutures  may  be 
sterilized  with  steam  without  injuring  them  except  catgut 
which  swells  up  and  softens  and  becomes  useless  when  treated 
in  this  way.  Catgut  is  of  such  great  value  as  a  suture  material 
particularly  for  deeply  buried  sutures  and  in  abdominal  sur- 
gery on  account  of  the  fact  that  the  catgut  does  not  have  to  be 
removed,  but  is  absorbed  by  the  tissues  that  every  effort  has 
been  made  to  devise  a  sure  method  for  its  sterilization.  Con- 
sisting as  is  does  of  the  animal  intestine  it  is  apt  to  contain 
pathogenic  bacteria  the  colon  bacillus  at  least  derived  from  the 
feces  with  which  it  was  in  contact,  and  is  to  be  regarded  as 
essentially  infectious  in  itself.  So  far  no  certain  means  of 
sterilization  have  been  devised  for  catgut,  though  very  many 
experiments  have  been  made  to  accomplish  it.  The  methods 
which  seem  to  have  geven  the  best  results  all  consist  in  sub- 


266  MANUAL    OF    BACTERIOLOGY. 

jecting  the  gut  to  high  temperatures  in  alcohol  or  some  heavy 
oil  which  does  not  materially  injure  its  structure.  Some  sur- 
geons, however,  have  reluctantly  abandoned  the  use  of  catgut 
altogether  on  account  of  the  difficulty  of  sterilizing  it.  Silver 
wire  is  a  valuable  mateiial  for  sutures  for  the  reason  that  it 
may  be  easily  sterilized  by  heat  without  injuring  it,  and  more- 
over silver  is  itself  a  potent  germicide.  Stitch  abscesses  sel 
dom  or  never  form  around  silver  wire  sutures. 

For  metallic  instruments  boiling  for  five  minutes  in  i  per  cent, 
soda  solution  is  said  to  suffice  amply  for  thorough  sterilization. 
This  does  not  dull  the  edge  provided  the  instruments  are  sup- 
ported on  some  kind  of  rack  above  the  bottom  of  the  vessel  in 
which  they  are  sterilized.  But  they  should  be  properly  con- 
structed in  the  first  place,  and  should  consist  entirely  of  metal. 
Scissors,  hemostats,  syringes  and  the  like  should  be  so  con- 
structed that  they  are  easily  taken  apart. 

Brushes  used  for  scouring  the  hands  and  the  field  of  opera 
tion  are  difficult  to  sterilize  with  certainty.     The  best  method 
seems  to  be  to  sterilize  them  in  the  autoclave  and  lay  them  in  a 
disinfecting  solution  till  needed. 

Rubber  catheters  are  very  hard  to  sterilize.  The  best  way 
seems  to  be  to  keep  them  in  corrosive  sublimate  solution  i-iooo, 
and  to  be  sure  that  the  disinfectant  fills  the  lumen  as  well  as 
coming  in  contact  with  the  outside. 

Sterilization  of  solutions  used  in  subcutaneous  injections  is 
attended  with  some  difficulty.  Some  of  the  chemicals  of  which 
these  solutions  are  made  are  injured  by  heat.  Various  sug- 
gestions have  been  made  to  render  such  injections  less  liable  to 
produce  infection.  Making  up  the  solutions  with  i-ioooo  cor- 
rosive sublimate  is  advised,  also  keeping  the  drugs  dissolved  in 
strong  alcoholic  solutions,  and  diluting  with  sterilized  water  as 
needed.  Various  infections  have  been  from  time  to  time  re- 
ported from  the  use  of  subcutaneous  injections  of  contaminated 
solutions. 


PART   111. 

NON-PATHOGENIC  BACTERIA. 

No  very  sharp  line  can  be  drawn  between  pathogenic  bacteria 
on  the  one  hand,  and  non-pathogenic  bacteria  on  the  other;  for 
many  kinds  of  bacteria  which  occur  as  saprophytes  under 
ordinary  circumstances  may  multiply  in  the  botly  and  cause 
injury  under  certain  conditions.  Bacillus  prodigiosus,  an  or- 
ganism which  is  given  to  students  in  the  beginning  of  their 
studies  in  bacteriology,  on  account  of  its  harmlessness,  pro- 
duces lesions  in  experiment  animals  when  injected  into  the 
peritoneal  cavity.  But  those  organisms  may  be  fairly  classed 
as  non-pathogenic  which  are  not  found  to  cause  spontaneous 
disease,  and  which  do  not  cause  disease  in  animals  when  in 
troduced  in  small  amounts. 

The  number  of  species  of  non-pathogenic  bacteria  is  very 
large.  Eisenberg*  describes  376  species  of  bacteria,  mostly 
non-pathogenic.  Sternbergf  enumerates  489  species,  includ- 
ing the  pathogenic  varieties;  but  the  majority,  of  course,  are 
nonpathogenic.  FliiggeJ  considers  about  500  species  of  bac- 
teria. Migula§  recognizes  nearly  1300,  and  Chester  ||  about 
800  species.  Probably  some  of  the  bacteria  which  have  been 
described  as  distinct  species  are  in  reality  not  different;  but,  on 
the  other  hand,  it  is  also  probable  that  a  still  larger  number  of 
spices  have  not  been  described  at  all — how  many,  it  is  impos- 

*Bakteriologische  Diagnostik.     1891. 

tManual  of  Bacteriology.     1893. 

%Die  Mikroorganismen.     1896. 

^System  der  Bakterien.     1900. 

[[Manual  of  Determinative  Bacteriology.     1901. 

267 


268  MANUAL    OF    BACTERIOLOGY. 

sible  to  say.  In  a  work  of  this  character  it  is  feasible  to  men- 
tion only  a  few  of  the  commonest  and  best-known  species 
of  non-pathogenic  bacteria  or  such  as  have  some  marked 
peculiarity. 

Micrococcus  Agilis. — Found  in  water;  coccus  about  i  ^  in 
diameter,  usually  appearing  as  diplococci,  sometimes  as  strepto- 
cocci and  tetrads;  liquefies  galatin  slowly;  grows  at  room  tem- 
perature, on  ordinary  culture  media,  forming  a  rose-red  pig- 
ment on  agar  and  potato.  This  micrococcus  is  remarkable 
in  being  actively  motile;  it  possesses  a  flagellum.  It  is  stained 
Gram's  method.  • 

Micrococcus  Ureae. — Found  in  decomposed,  ammoniacal 
urine  and  in  the  air;  coccus,  0.8  to  i  ^  in  diameter,  occurring 
singly  or  in  various  combinations;  does  not  liquefy  gelatin; 
facultative  anaerobic;  grows  rapidly,  best  at  30°  to  33°  C. ; 
grows  on  ordinary  gelatin,  but  best  on  special  media;  it  de- 
composes urea,  producing  ammonia  and  carbon  dioxide,  which 
form  ammonium  carbonate. 

Sarcinae. — There  is  a  large  number  of  species  of  sarcinae. 
They  are  common  organisms  in  the  air.  They  frequently  con- 
taminate plate-cultures.  Many  of  the  sarcinae  of  the  air  pre- 
sent, in  cultures,  growths  having  brilliant  colors,  from  which 
some  of  them  are  named;  thus  there  are  arange,  yellow,  rose- 
colored  and  white  sarcinae,  and  others. 

Sarcina  Pulmonum.— Found  in  the  air-passages  of  man; 
i  to  1.5  M  in  diameter,  occurring  in  tetrads  or  cubes  of  eight 
cells;  aerobic;  does  not  liquefy  gelatin;  grows  slowly,  best  at 
ordinary  temperature,  preferably  upon  gelatin.  It  decom- 
poses urine  with  the  formation  of  ammonia.  It  is  said  to  form 
endogenous  spores  which  are  extremely  resistant  toheat. 

Sarcina  Ventriculi.— Found  in  the  stomachs  of  man  and 
of  animals;  2.5  M  in  diameter,  occurring  in  cubes  of  eight  cells 
or  more;  it  does  not  liquefy  gelatin;  aerobic:  grows  on  ordinary 
culture-media;  the  growths  tend  to  become  yellow.  Small 


NON-PATHOGENIC   BACTERIA.  269 

numbers  of  sarcinae  may  occur  in  the  normal  human  stomach; 
the  presence  of  large  numbers  indicates  the  existence  of  ab- 
normal fermentative  processes. 

Bacillus  Fluorescens  Liquefaciens.— Found  in  water  and 
putrid  fluids;  very  common;  appears  as  a  small  rod,  actively 
motile;  aerobic,  but  somewhat  variably;  liquefies  gelatin; 
grows  rapidly  at  ordinary  temperatures  upon  the  usual  cul- 
ture-media. It  forms  a  pigment  having  a  beautiful  greenish- 
yellow  fluorescence,  best  seen  in  transparent  media;  the 
growth  on  potato  has  a  brown  color.  Does  not  stain  by  Gram's 
method  and  does  not  form  spores. 

Bacillus  Fluorescens  Putidus.— Found  in  water;  a  short 
rod  with  rounded  ends;  actively  motile;  does  not  liquefy  gelatin; 
aerobic;  does  not  form  spores;  grows  rapidly  at  the  ordinary 
temperatures  upon  the  common  media.  Gelatin  cultures  give 
off  a  powerful,  foul  odor  of  trimethylamin.  It  produces  a 
greenish,  fluorescent  pigment,  best  seen  in  transparent  media; 
on  potato  the  growths  form  a  thin,  gray  to  brown,  slimy  layer. 

There  are  several  other  fluorescing  bacilli,  mostly  found  in 
water. 

Bacillus  Indicus.— Found  by  Koch  in  the  stomach  contents 
of  an  ape  in  India;  a  fine  short  bacillus  with  rounded  ends; 
motile;  does  not  form  spores;  facultative  anaerobic;  liquefies 
gelatin;  grows  rapidly,  best  at  35°  C.  upon  the  ordinary  media; 
produces  a  brick-red  pigment.  Very  large  doses  injected  into 
rabbits  caused  death  in  three  to  twenty-four  hours. 

Bacillus  Prodigiosus. — Widely  disseminated  in  the  atmos- 
phere of  certain  places;  a  short  bacillus  with  rounded  ends, 
in  form  often  nearly  like  the  micrococci;  facultative  anaerobic; 
not  motile,  as  a  rule;  does  not  form  spores;  liquefies  gelatin 
rapidly;  grows  rapidly,  best  at  25°  C.  on  the  ordinary  culture- 
media;  milk  is  coagulated;  gas  forms  in  sugar-media;  cultures 
on  potatoes  give  off  a  foul  odor  of  trimethylamin.  A  brilliant 
red  color,  which  develops  only  in  the  presence  of  oxygen,  ap- 


270  MANUAL    OF    BACTERIOLOGY. 

pears  in  cultures.     The  pigment  appears  as  granules  outside 
of  the  bacteria. 

Bacillus  Violaceus  (of  Berlin)'.— Found  in  water;  a  slim 
rod  with  rounded  ends  which  may  form  threads;  actively 
motile;  facultative  anaerobic;  liquefies  gelatin  rapidly;  forms 
endogenous  spores  placed  near  the  centers  of  the  bacilli;  grows 
rapidly,  and  not  at  high  temperatures,  upon  ordinary  media, 
forming  a  deep,  violet-colored  pigment.  There  are  several 
bacilli  related  to  this  one. 

Bacillus  Amylobacter  (Clostridium  butyricum;  Bacillus 
butyricus,  Prazmowski). — Found  widely  distributed  in  nature 
in  decomposing  vegetable  material  and  in  the  stomachs  of 
ruminant  animals;  a  large,  thick  rod  with  round  ends,  often 
arranged  in  chains;  actively  motile;  anaerobic;  forms  spores 
which  are  located  in  the  center  of  the  bacillus  and  give  it  a 
spindle-shaped  form,  or  at  one  end,  when  it  has  the  outline 
of  a  tadpole;  has  not  been  cultivated  satisfactorily  on  ordinary 
media;  grows  best  at  35°  to  40°  C.;  decomposes  carbohydrates 
with  the  formation  of  butyric  acid;  decomposes  cellulose. 
Organisms  of  similar  form  have  been  found  as  fossils  belonging 
to  the  carboniferous  period. 

Bacillus  Butyricus  (Hueppe).— Found  in  milk;  appears  as 
a  small,  irregular  rod,  also  forming  threads;  very  actively  motile; 
aerobic;  rapidly  liquefies  gelatin;  forms  centrally  located  spores; 
grows  best  at  35°  to  40  C.;  grows  rapidly  on  ordinary  media; 
coagulates  milk,  redissolving  the  coagulum,  producing  also 
butyric  acid.  A  large  number  of  bacteria,  both  aerobic  and 
anaerobic,  produce  butyric  acid  fermentation. 

Bacillus  Megaterium.— Obtained  by  de  Bary  from  cooked 
cabbage-leaves;  common  on  plants  and  earth;  a  large  bacillus 
with  rounded  ends,  often  forming  chains;  motile;  slowly  lique 
fies  gelatin;  aerobic;  forms  spores,  especially  in  potato  cul- 
tures; grows  rapidly  at  room  temperature  on  the  ordinary 
media. 


NON- PATHOGENIC    BACTERIA.  271 

Bacillus  Mesentericus  Vulgatus  (Potato bacillus).— Found 
on  potatoes;  common  in  earth;  a  large,  long  rod  with  rounded 
ends,  often  forming  long  chains;  motile;  it  is  stained  by  Gram's 
method;  liquefies  gelatin;  aerobic;  forms  spores;  grows  rap- 
idly, best  at  about  20°  C.;  grows  on  ordinary  media,  forming 
on  potato  a  thin,  wrinkled  membrane  which  spreads  rapidly 
over  the  surface.  It  coagulates  milk,  redissolving  the  coag- 
ulum.  It  possesses  numerous  flagella.  The  spores  are  ex- 
tremely resistant  to  heat. 

Bacillus  Phosphorescens  Indicus.— Obtained  from  sea- 
water;  a  small,  thick,  rod-shaped  bacillus  with  rounded  ends, 
also  forming  threads;  actively  motile;  not  stained  by  Gram's 
method;  liquefies  gelatin;  aerobic.  It  grows  slowly,  best  be- 
tween 20°  and  30°  C.,  upon  the  usual  media,  except  milk  and 
potato.  Its  culture,  when  old,  especially  when  on  animal 
nutrient-media  and  in  the  presence  of  certain  sodium  salts, 
are  phosphorescent  in  the  dark. 

There  are  various  other  bacilli  which  produce  phosphores- 
cence, some  of  which  do  not  liquefy  gelatin. 

Bacillus  Mycoides  (Bacillus  ramosus;  Wurzelbacillus) .— 
Found  in  the  earth  and  in  water;  very  common;  a  large,  short 
bacillus  with  rounded  ends,  often  forming  chains  and  threads; 
slightly  motile;  liquefies  gelatin;  aerobic  forms  centrally  lo- 
cated, oval  spores;  grows  rapidly  at  room  and  incubator  tem- 
peratures upon  the  usual  media.  It  is  said  rapidly  to  decom- 
pose albumin  with  the  formation  of  ammonia. 

Bacillus  Subtilis  (Hay  bacillus).— Found  on  hay,  in  the 
air,  water,  ground  and  decomposing  fluids;  very  common; 
a  large  bacillus  somewhat  resembling  the  anthrax  bacillus  in 
form,  with  rounded  ends*  often  forming  chains  or  long  fila- 
ments; motile;  possessing  flagella;  liquefies  gelatin;  aerobic; 
it  is  stained  by  Gram's  method.  It  may  have  large,  centrally 
located  spores,  which  form  best  on  potato  at  about  30°  C. 
The  spores  are  extremely  resistant  to  heat  and  to  chemical 


272  MANUAL   OF    BACTERIOLOGY. 

germicides.  It  grows  best  at  about  30°  C.  upon  the  ordinary 
culture- media;  milk  is  peptonized.  Bacillus  subtilis  may 
easily  be  isolated  in  pure  culture  by  adding  finely  cut  hay  to 
tubes  of  bouillon;  placing  these  in  the  steam  sterilizer  for  five 
or  ten  minutes;  then  letting  the  tubes  develop  in  the  incubator. 
Plates  made  from  the  bouillon  will  probably  show  colonies  of 
the  Bacillus  only,  as  the  steam  may  be  expected  to  have  de- 
stroyed all  organisms  except  its  very  resistant  spores. 


FIG.  59. — Bacillus  subtilis.     (X  1000.) 

The  hay  bacillus  has  certain  congruers,  and  it  is  perhaps 
more  correct  to  speak,  as  is  often  done,  of  the  "hay  bacillus 
group"  rather  than  of  a  special  organism.  Some  of  the  con- 
gruers have  been  found  in  pure  culture  in  cases  of  panophthal- 
mitis  following  injury.  Moreover,  injections  of  cultures  of 
the  organism  so  obtained,  produced  panophthalmitis  in  experi- 
ment animals.* 

*Silberschmidt  Ann.  de  F  Institute  Pasteur.  1903.  p.  268.  Also  see 
Kneass  and  Sailer.  Univer.  Pennsylvania  Med.  Bull.  June,  1903. 


NO N- PATHOGENIC    BACTERIA.  273 

Bacillus  Erythrosporus.— Found  in  decomposing  fluids 
and  water;  a  slim  bacillus  with  rounded  ends;  motile;  does  not 
liquefy  gelatin;  facultative  anaerobic;  forms  oval,  red-colored 
spores,  two  to  eight  in  each  filament;  grows  rapidly,  only  at 
ordinary  temperatures;  produces  a  greenish-yellow,  fluorescent 
pigment.  On  potato  it  forms  a  limited,  reddish  growth  be- 
coming nut-brown. 

Bacillus  Cyanogenus  (Bacterium  syncyanum;  Bacillus 
lactis  cyanogenus;  Bacillus  of  blue  milk). — A  bacillus  of  vari- 
able size,  with  rounded  ends;  motile;  spore  formation  doubtful; 
is  aerobic;  not  stained  by  Gram's  method;  grows  rapidly  at 
ordinary  but  not  so  well  at  incubator  temperatures  on  the  usual 
culture-media;  does  not  liquefy  gelatin;  produces  a  grayish- 
blue  pigment,  brighter  in  acid  media,  at  ordinary  tempera- 
tures; milk  is  not  coagulated  or  rendered  acid. 

Bacillus  Acidi  Lactici  (Hueppe).— Found  in  sour  milk;  a 
short,  plump  rod;  not  motile;  does  not  liquefy  gelatin;  facul- 
tative anaerobic;  grows  on  the  ordinary  media;  in  milk  causes 
development  of  lactic  acid  with  precipitation  of  casein  and 
production  of  gas  and  alcohol.  It  belongs  in  the  same  group 
as  B.  coli  communis  and  B.  lactis  aerogenes  (see  Part  IV.). 

There  are  numerous  other  bacteria,  such  as  the  Bacterium 
acidi  lactici,  which  cause  the  formation  of  lactic  acid  in  milk. 

Heinemann,*  as  the  result  of  his  studies,  comes  to  the  con- 
clusion that  it  is  not  justifiable  to  regard  B.  acidi  lactici  as  a 
specific  bacillus. 

Bacterium  Ureae. — A  short,  thick  bacillus  with  rounded 
ends;  not  motile;  aerobic;  found  in  ammoniacal  urine;  grows 
slowly  at  room  temperature  upon  gelatin,  which  is  not  lique- 
fied; decomposes  urea;  forms  ammonium  carbonate. 

Bacterium  Zopfii.— Found  in  the  intestines  of  hens,  in 
water  and  in  fecal  matter;  a  bacillus  0.75  to  i  Abroad  and  2  to  5 
A*  long;  may  form  threads.  Actively  motile;  does  not  liquefy 

*Heinemann.     Jour.  In}.  Diseases.     Vol.  III.,    p.   173. 
18 


274  MANUAL    OF    BACTERIOLOGY. 

gelatin;  aerobic;  involution  forms  are  often  seen  and  they  have 
been  described  as  spores;  grows  rapidly,  best  at  20°  C.  upon 
gelatin;  forms  branching  zooglceae.  It  is  a  member  of  the  same 
group  as  B.  proteus  (see  Part  IV.). 

Spirillum  Rubrum. — Found  by  Esmarch  in  the  putrefying 
cadaver  of  a  mouse;  short  spirals  twice  the  breadth  of  the 
cholera  spirillum,  usually  with  one  to  three  turns;  in  bouillon 
growing  into  long  spirals;  motile,  with  flagella;  spore  forma- 
tion doubtful;  facultative  anaerobic;  does  not  liquefy  gelatin; 
grows  slowly,  best  at  about  37°  C.  on  the  ordinary  media;  pro- 
duces a  wine-red  pigment  only  when  the  air  is  excluded. 

Spirillum  or  Spirochaeta  Dentium.— Found  in  the  mouths 
of  healthy  persons,  on  the  margins  of  the  gums  when  they  are 
covered  with  a  dirty  deposit;  long  spirals  with  several  wind- 
ings, uneven  in  thickness;  has  not  been  cultivated. 

Spirillum  Sputigenum.— Found  in  the  human  mouth  in 
healthy  persons  at  the  margin  of  the  gums;  curved  rods  or 
short  spirals  which  resemble  the  spirillum  of  cholera  in  form; 
has  not  been  cultivated. 

Spirillum  Rugula  (Vibrio  rugula). — Found  in  swamp  water, 
in  fecal  matter  and  in  the  tartar  of  the  teeth;  a  curved  rod 
0.5  to  2.5  M  broad  and  6  to  8  /*  long,  having  one  flat  spiral  wind- 
ing; motile,  with  flagella  at  the  ends;  probably  anaerobic; 
forms  spores  located  at  the  ends. 

Spirillum  Volutans. — Found  in  swamp  water;  very  long 
spirals  with  several  turns;  1.5  to  2  /*  broad  and  25  to  30  /*  long; 
motile,  with  a  flagellum  at  each  extremity.  The  protoplasm 
is  granular. 

Spirillum  Undula.— Found  in  putrefying  infusions  con- 
taining organic  matters;  a  rather  short  spiral  form  with  three 
turns  or  less,  about  i  /*  thick  and  8  to  12  M  long;  actively  motile, 
with  a  tuft  of  flagella  at  each  extremity;  has  been  cultivated  on 
agar. 

Spirillum    or  Spirochaeta  Plicatile.— Found    in  swamp 


NON-PATHOGENIC    BACTERIA.  275 

water;  spiral  forms  of  various  lengths;  sometimes  100  to  200  M, 
long;  actively  motile. 

The  spirilla  (vibrios  or  comma-shaped  forms)  closely  re- 
sembling the  spirillum  of  cholera,  will  be  considered  in  con- 
nection with  that  organism. 

Fusiform  Bacillus. — Vincent*  was  the  first  to  describe 
fusiform  bacilli  which  he  isolated  from  a  case  of  diphtheroid 
angina,  and  since  his  publication  his  observations  have  been 
more  or  less  corroborated  by  a  number  of  others.  , 


\ 

c 


FIG.  60. — Spirilla  from  swamp  water.     (X  about  500.) 

Weaver  and  Tunniclifff  cultivated  fusiform  bacilli  from  a 
case  of  ulceromembranous  stomatitis  and  from  a  case  of 
diphtheria.  The  cultures  were  grown  under  anaerobic  condi- 
tions at  37°  C.  They  were  inclined  to  regaid  the  spiral  forms 
always  present  along  with  the  fusiform  organism  as  different 
from  these,  but  upon  fuithei  observation  TunnicliffJ  showed 

*Ann.  de  Vlnst.  Pasteur.     1899.     p.  609.     (Cited  from  Giinther.       Loc.  cit.) 
t  Weaver  and  Tunnicliff.     Journal  Infectious  Diseases.     Vol.  II.,  1905.     pp 
446-459.     Also  Ebenda.     Vol.  IV.,  No.     i.  pp.  8-33. 

tTunnicliff.     Journ.  Infectious  Diseases.     Vol.  III.,  1906.     pp.  148-155. 


276  MANUAL    OF    BACTERIOLOGY. 

that  the  spiral  forms  are  in  reality  not  separate  organisms  but 
represent  stages  in  the  cycle  of  development  of  the  fusiform 
bacillus  which  is  polymorphous  to  a  very  marked  degree, 
showing  short  bacilli,  long  filaments  and  spirals.  The  bacilli 
form  spores,  usually  one  spore  to  each  bacillus,  sometimes  two. 
The  filaments  are  either  straight  or  wavy.  The  spores  retain 
the  stain  when  treated  as  in  staining  tubercle  bacilli  and  de- 
colorized with  i  per  cent,  sulphuric  acid.  The  cultures  upon 


c 


TIG.  61. — Spirilla  from  swamp  water  showing  flagella  (Loffler 
stain).     (X 1000.) 

which  Tunnicliff's  observations  were  made  were  obtained  from 
three  normal  throats,  but  the  culture  corresponded  in  all 
respects  with  those  obtained  by  her  and  Weaver  already 
mentioned. 

Higher  Bacteria. — Certain  organisms  (beggiatoa,  thiothrix 
leptothrix,  cladothrix,  actinomyc.es  or  streptothrix)  of  more 
complicated  structure  than  most  bacteria,  but  resembling  them 
in  many  respects,  are  called  "higher  bacteria."  They  consist 
of  definite  filaments  which  are  usually  made  up  of  rod-shaped 
elements,  but  the  relation  between  these  elements  is  very  intimate. 


NON-PATHOGENIC    BACTERIA.  277 

Some  of  them  (beggiatoa,  thiothrix)  contain  sulphur  granules. 
Many  of  them  occur  in  water.  There  are  foims  among  them 
which  are  found  attached  to  some  object  by  one  end  of  the  fila- 
ment (thiothrix).  Some  of  them  (actinomyces  or  streptothrix) 
have  branching  filaments,  which  are  rarely  seen  among  the 
lower  bacteria  (see  page  119).  Often  one  end  of  the  filament 
becomes  specialized  for  the  purposes  of  reproduction.  The 
fungus  of  actinomycosis  is  the  best  known  of  this  group. 
There  are  many  other  members,  however,  both  pathogenic 
and  non-pathogenic.  Most  of  them  require  still  further  study. 
The  tubercle  bacillus  and  other  acid-proof  bacilli  which  re- 
semble it  have  some  points  of  resemblance  with  actinomyces 
(see  B.  tuberculosis,  Part  IV.). 

Leptothric  Buccalis.— Found  in  the  mouth  cavity.  This 
name  has  been  applied  to  large,  twisted,  thread-like  organisms, 
in  which  segments  can  be  demonstrated  with  difficulty  or  not 
at  all.  Apparently,  different  organisms  have  been  described 
under  this  name.  Vignal  claims  to  have  cultivated  a  Lepto- 
thrix  buccalis.  Miller  recognizes  two  principal  species,  neither 
of  which  could  be  cultivated — Leptothrix  innominata,  which 
shows  no  transverse  divisions,  and  which  is  stained  faintly 
yellow  by  iodine;  and  Bacillus  buccalis  maocimus,  in  which  the 
transverse  divisions  are  distinct,  and  which  is  stained  brown- 
ish-violet by  iodine.  Miller's  Leptothrix  maxima  buccalis  is 
similar  to  the  last  except  in  lacking  the  iodine  reaction. 

A  variety  of  leptothrix,  or  a  nearly  related  organism,  appears 
to  be  the  most  frequent  cause  of  the  form  of  gangrenous  in- 
flammation of  the  mouth  and  genitals  called  noma.  It  stains 
faintly  by  Gram's  method.  It  does  not  grow  on  ordinary 
media.*  Another  organism  of  this  group  has  been  described 
which  is  pathogenic  to  a  number  of  domestic  animals. f 

*Blumer  and  MacFarlane.  American  Journal  Medical  Sciences.  November, 
1901. 

fit  has  also  been  called  "necrosis  bacillus,"  and  "Streptothrix  cuniculi." 
Pearce.  University  of  Pennsylvania  Medical  Bulletin.  November,  1902. 


278  MANUAL    OF    BACTERIOLOGY. 

Yeasts  and  Moulds. — In  the  course  of  bacteriological  work 
one  constantly  encounters  yeasts  and  moulds,  which,  although 
not  bacteria,  must  nevertheless  be  understood  and  recognized 
to  avoid  error.  Accidental  contamination  of  tubes  or  plates 
is  likely  to  be  the  result  of  the  growth  of  some  of  these  forms. 
The  yeasts  generally  go  by  the  name  of  saccharomyces,  of  which 
there  are  several  spices.  The  Saccharomyces  cerevisia  is  the 


FIG.  62. — Yeast  cells  stained  with  fuchsin.     (X  1000.) 

ordinary  yeast  of  alcoholic  fermentation.  Some  of  the  yeasts 
present  colored  growths — red,  white  and  black.  They  con- 
sist of  large,  oval  cells,  which  readily  stain  with  the  aniline  dyes. 
They  multiply  by  the  protrusion  of  a  little  bud  from  the  cell, 
which  develops  into  a  new  cell.  In  an  actively  germinating 
growth  of  yeast  these  budding  cells  are  readily  distinguished 
(Fig.  62). 

Among  the  moulds  the  varieties  most  commonly  encountered 
are  the  mucor,  the  penicillium,  the  aspergillus  and  the  oidium. 


NON-PATHOGENIC    BACTERIA. 


279 


FIG.  63. — (Baumgarten.} 

a.  Penicillium  glaucum.     b.  Oiidium  lactis.     c.  Aspergillus  glaucus.     d.  The 
same  more  highly  magnified,     e.  Mucor  mucedo. 


28o  MANUAL   OF    BACTERIOLOGY. 

There  are  various  species  of  each  of  them.  They  consist  of 
cells  arranged  end  to  end,  making  a  thread-like  body  called  a 
hypha.  The  threads  are  matted  together  and  form  a  mycelium. 
Certain  threads  project  upward  from  the  mycelium,  and  on 
them  are  borne  spores.  The  arrangement  of  the  spores  is 
characteristic  in  each  variety  of  mould  (Fig.  63).  A  group  of 
organisms  exist  which  have  affinities  both  with  yeasts  and 
mould-fungi.  Some  of  them  are  pathogenic.  The  form  of 
infection  of  the  mouth  called  thrush  is  due  to  a  fungus  of  this 
class,  which  is  generally  considered  an  oi'dium.  A  chronic  in- 
flammatory affection  of  the  skin  (blastomycetic  drematitis)  is 
due  to  related  organisms.*  Irons  and  Graham \  have  reported 
a  case  of  generalized  blastomycosis  inwhich  they  isolated  from 
the  several  lesions  during  life  and  post-mortem  an  organism 
corresponding  with  oidium  as  described  by  Ricketts.  Others 
have  reported  similar  cases,  but  with  more  or  less  reserve  as 
to  the  etiological  significance  of  the  organism.  Irons  and 
Graham  excluded  the  tubercle  bacillus,  and  furthermore  ob- 
tained positive  results  on  the  inoculation  of  animals,  and  they 
regard  the  organism  as  the  cause  of  the  disease  in  the  case 
reported. 

LeCount  and  Meyers  J  report  a  case  of  systemic  blastomy- 
cosis in  which  there  were  foci  of  infection  very  generally  distrib- 
uted throughout  the  body,  involving  not  only  the  abdominal 
and  thoracic  viscera,  but  also  the  cerebellum,  and  the  left  el- 
bow and  both  knee  and  ankle-joints. 

Hamburger§  made  a  study  of  the  organisms  derived  from  four 
cases  reported  respectively  by  ||Bassoe,  Irons  and  Graham, If 

*Ricketts.  Journal  of  Medical  Research.  Vol.  VI.,  1901.  Hyde  and 
Montgomery.  Journal  American  Medical  Association.  Tune  7.  1002.  Ophiils. 
Journ.  Exper.  Med.  VI. 

"fjour.  Infectious  Diseases.     Vol.  III.,  pp.  443-682.     1906. 

JLeCount  and  Meyers.     Journ.  Inf.  Dis.     Vol.  IV.,  1907.     pp.  187-200. 

§Journ.  Infec.  Dis.     Vol.  IV.,  1907.     pp.  201-209. 

||  Journ.    Infect.    Dis.    1906.     p.    91. 

^[   Loc.    cit.  supra. 


NON-PATHOGENIC    BACTERIA.  281 

and  Christensen  and  Hektoen,*  the  last  two  having  reported 
two  of  the  cases.  Hamburger  found  the  four  strains  to  be 
nearly  identical.  They  grow  on  all  the  ordinary  culture  media, 
best  perhaps  on  slightly  acid  glucose  media.  In  cultures  from 
case  No.  2  budding  forms  were  not  observed.  Gross  and 
microscopical  differences  in  the  organisms  are  produced  by 
varying  temperatures. 

The  sporotricha  of  Schenckf  which  produces  chronic  sub- 
cutaneous abscesses,  ma'y  be  mentioned  here,  provisionally.  A 
number  of  skin  affections,  such  as  tinea  favosa  and  tinea 
trichophytina,  are  due  to  fungi,  which  have  some  similarity  to 
those  above  mentioned. 

Among  the  mould  fungi,  several  species  of  aspergillus  and  of 
mucor  are  pathogenic.  Man,  as  well  as  the  lower  animals, 
may  be  affected.  In  man  the  lungs  may  be  involved  in  a 
broncho-pneumonia  (pneumonomycosis),  usually  due  to  asper- 
gillus, and  often  secondary  to  some  preexisting  disease  of  the 
lung.  Mould  fungi,  especially  aspergillus,  may  grow  in  the 
external  ear  (otomycosis) .  The  growth  is  usually  superficial. 
These  fungi  rarely  produce  lesions  in  other  organs. 

*Journ.  Am.  Med.  Assn.  1906.     p.  247. 

fHektoen.     Journal  Experimental  Medicine.     Vol.  V. 


PART  IV. 

PATHOGENIC  BACTERIA. 

Suppuration  and  Allied  Conditions. — The  occurrence  of 
suppuration  is  characterized  by  certain  appearances  which  we 
are  accustomed  to  describe  under  the  name  of  inflammation. 
The  study  of  inflammation  belongs  to  pathology,  and  cannot 
be  considered  fully  here.  However,  certain  evidences  which 
are  characteristic  of  the  suppurative  variety  of  inflammation 
need  to  be  outlined  on  account  of  their  relation  to  the  action  of 
the  pyogenic  bacteria. 

In  a  suppurating  area,  as  is  well  known,  the  blood-vessels 
are  dilated,  and  the  lymph-spaces  become  filled  with  serum. 
Leukocytes  are  attracted  to  the  neighborhood  in  large  num- 
bers, by  positive  chemotaxis,  and  crowd  the  small  veins  and 
capillaries.  The  leukocytes,  by  reason  of  their  ameboid  move- 
ment, pass  through  the  walls  of  the  vessels  at  little  openings 
filled  with  cement-substance,  situated  between  the  lining  endo- 
thelial  cells.  According  to  the  theory  of  phagocytosis,  they  are 
bent  on  finding  the  irritant  which  has  led  to  the  inflammation, 
and  upon  attacking  it  and  rendering  it  harmless.  At  the  point 
which  appears  to  be  the  center  of  the  inflammatory  area  there 
is  usually,  but  not  always,  a  necrosis  of  the  cells  of  the  tissue; 
this  constitutes  the  central  slough  or  the  familiar  core  found  in 
some  boils.  The  necrosis  is  to  be  attributed  to  poisons  formed 
by  the  micrococci.  In  sections  cut  through  such  an  abscess 
the  nuclei  of  the  necrotic  cells  in  the  center  fail  to  take  the 
nuclear  stain ;  the  necrotic  mass  does  not  stain,  or  takes  the  dye 
diffusely  and  irregularly,  and  it  is  broken  up  into  fine  granules. 

282 


PATHOGENIC    BRCTERIA.  283 

The  cells  of  the  tissues  surrounding  the  necrotic  area  are 
mingled  with  large  numbers  of  polynuclear  leukocytes,  which 
enclose  the  area  of  irritation. 

The  nuclei  of  the  cells  near  the  center  of  the  abscess  are  fre- 
quently broken  up  into  a  number  of  small  fragments,  which  in 
dicates  the  commencement  of  their  destruction.  In  sections 
through  small  abscesses  it  is  possible,  by  means  of  a  double 
stain  of  carmine,  followed  by  Gram's  method,  to  bring  out 
the  histological  character  of  the  tissue,  and  at  the  same  time 
to  stain  the  common  phyogenic  bacteria,  which  are  usually 
found  near  the  center  of  the  abscess  in  large  numbers,  even 
making  masses  visible  with  a  low  power  of  the  microscope. 
It  is  often  possible  by  this  method  to  demonstrate  masses  of 
micrococci  filling  up  the  lumina  of  capillaries  in  which  they 
are  lodged  as  emboli. 

The  production  of  pus  in  the  center  of  the  abscess  is  due  to 
the  liquefaction  of  the  necrotic  tissue,  which  apparently  results 
from  the  action  of  some  peptonizing  ferment.  In  the  liquid 
thus  formed  immense  numbers  of  the  polynuclear  leukocytes 
are  found  floating,  and  they  constitute  the  greater  part  of  the 
so-called  pus-cells.  The  nuclei  of  these  cells  are  obscured  by 
clouds  of  extremely  fine  granules.  The  granules  are  of  an 
albuminoid  nature,  and  are  dissolved  by  acetic  acid,  when  the 
nuclei  become  visible.  The  nuclei  generally  consist  of  three, 
four,  or  five  more  portions.  Pus-cells  may  contain  fatty 
granules;  sometimes  the  cells  are  necrotic;  sometimes  living 
leukocytes  may  be  present.  The  pus-cells  may  also  contain 
bacteria.  The  presence  of  the  fine  albuminoid  granules  in  the 
pus-cells  is  to  be  counted  as  a  degenerative  change.  Although 
it  is  possible  to  produce  suppuration  experimentally  by  the 
introduction  of  sterilized  irritants,  such  as  croton  oil,  into  the 
tissues  of  animals,  in  all  cases  met  with  in  practice  suppuration 
is  due  to  the  action  of  pyogenic  bacteria. 

Specimens  of  pus  will  nearly  always  be  found  to  contain 


284  MANUEL   OF    BACTERIOLOGY. 

bacteria,  which  can  be  demonstrated  by  cultivation,  and,  as  a 
rule,  also  in  smears  made  and  stained  upon  cover-glasses. 
The  bacteria  in  ordinary  suppuration  lie,  for  the  most  part,  out- 
side the  pus-cells,  though  some  of  them  maybe  found  in  the  pus- 
cells.  In  the  case  of  the  gonococcus  and  the  Diplococcus  in- 
tracellularis  meningitidis  they  are  characteristically  found  in 
pairs,  inside  of,  or  at  least  attached  to,  the  pus-cells.  The 
character  of  the  suppuration  differs  somewhat  with  the  different 
species  of  pyogenic  bacteria.  The  kind  of  abscess  above  de- 
scribed—localized and  having  a  central  slought,  usually  rather 
slow  in  progress — is  typical  for  the  Staphylococcus  pyogenes 
aureus,  which  is  prone  to  produce  circumscribed  areas  of  sup- 
puration. The  Sterptococcus  pyogenes,  on  the  other  hand, 
of tener  leads  to  suppuration  of  a  more  diffused  character,  such  as 
we  see  in  cellulitis  and  erysipelas;  but  either  organism  may,  at 
times,  produce  the  effects  usually  characteristic  of  the  other. 
Pus  having  a  blue  or  green  tinge  generally  owes  the  color  to 
the  presence  of  the  Bacillus  pyocyaneus.  The  commonest 
pus  producing  organism  is  then  the  Staphylococcus  pyogenes 
aureus,  and  next  to  that  the  Streptococcus  pyogenes.  Among 
the  other  pyogenic  bacteria  the  following  may  be  named: 

Staphylococcus  pyogenes  albus,  including  Staphylococcus 
epidermidis  albus;  streptococcus  of  erysipelas  (probably  iden- 
tical with  Streptococcus  pyogenes);  gonococcus;  Diplococcus 
mtracellularis  meningitidis;  Staphylococcus  pyogenes  citreus; 
Micrococcus  tetragenus;  Micrococcus  pyogenes  tenuis,  which 
may  be  the  same  as  the  Micrococcus  lanceolatus;  Staphy- 
lococcus cereus  albus  and  flavus. 

Pus-formation  may  also  be  due  to  Micrococcus  lanceolatus, 
Bacillus  pyocyaneus,  Bacillus  proteus,  Bacillus  coli  communis, 
Bacillus  pyogenes  fcetidus,  Bacillus  pneumonia  (of  Friedlander), 
Bacillus  aerogenes  capsulatus,  the  ray  fungus  of  actinomycosis, 
and  possibly  the  bacillus  of  bubonic  plague.  Besides  these 
organisms,  there  are  others  whose  effects  are  usually  more 


PATHOGENIC    BACTERIA. 


285 


marked  in  a  specific  way  which  sometimes  form  pus,  as  the 
bacilli  of  diphtheria,  tuberculosis,  glanders  and  typhoid  fever. 
Frequently  two  or  more  species  of  pyogenic  bacteria  will  be 
found  associated. 

The  table  below,  quoted  from  Dowd,  shows  the  frequency 
of  the  occurrence  of  various  pyogenic  bacteria  in  135  cases  of 
different  types  of  suppuration. 


Cellulitis,  51  cases. 

Infected  fresh  wounds, 
17  cases. 

Old  granulating  wounds, 
1  8  cases. 

Healing  wounds:  stitches, 
5  cases. 

i 

o 

^0) 

fc 

Abscesses,  37  cases. 

Streptococcus  pyogenes  alone 

Streptococcus  pyogenes  predominant  

2T. 

T. 

8 

Streptococcus  pyogenes  relatively  few  ..... 

T. 

I 

6 

I 

Staphylococcus  aureus  alone 

1  1 

I 

I 

I 

7 

6 

Staphylococcus  pyogenes    aureus  predom- 
inant 

8 

2 

i 

Staphylococcus  pyogenes  aureus  relatively 
few 

1  3 

•} 

2 

Staphylococcus  pyogenes    or    epidermidis 
albus  alone 

i 

4' 

2 

4 

2 

Staphylococcus  pyogenes    or    epidermidis 
albus  predominant                .        

I 

Staphylococcus  pyogenes    or    epidermidis 

10 

6 

2 

I 

2 

I 

II 

5 

? 

Bacillus  pyocycineus 

X 

3 

Bacillus  coli  communis 

3 

O  vergro  wn 

2 

i 

2 

e 

The  condition  of  the  animal's  tissues  is  of  great  importance 
in  determining  whether  or  not  suppuration  will  occur  when 
they  are  exposed  to  infection.  It  will  be  seen  that  we  are  re- 
peatedly subjected  to  infection  with  pyogenic  bacteria,  but  that 


286  MANUAL    OF    BACTERIOLOGY. 

in  most  cases  suppuration  nevertheless  does  not  occur.  The 
local  conditions  have  an  important  influence  in  determining 
infection.  Regions  of  hyperemia,  edema,  anemia  or  necrosis 
are  especially  liable  to  suppuration,  as  are  tissues  which  have 
been  bruised,  lacerated,  strangulated  or  otherwise  damaged. 
Furthermore,  the  general  condi;ion  of  the  patient  is  of  great  im- 
portance. Chronic  diseases  and  conditions  of  exhaustion  or 
depression  dispose  to  suppuration,  and  the  depraved  condi- 
tion of  the  tissues  in  diabetes  renders  the  sufferei  from  this 
disease  especially  liable  to  it.  These  facts  have  already  been 
enumerated  in  a  previous  chapter  (page  178).  In  the  lower 
animals  we  find  that  it  is  often  very  difficult  to  produce  sup- 
puration artificially  with  the  ordinary  pyogenic  bacteria.  In 
rabbits  the  subcutaneous  introduction  of  Staphylococcus  pyo- 
genes  aureus  frequently  fails  to  produce  an  abscess.  Suppura- 
tion is  likely  to  result,  however,  if  an  irritant  body  like  a  piece 
of  sterilized  potato  or  sterilized  glass  be  introduced  along  with 
the  bacteria. 

There  are  probably  a  number  of  other  specific  predisposing 
causes  in  the  animal  body  about  which  we  are  only  beginning 
to  obtain  an  understanding.  The  weakening  of  the  alexins, 
the  absence  of  opsonins  and  other  intricate  conditions  are 
probably  subject  to  great  variability,  and  may  serve  to  explain 
the  tendency  to  infection  at  certain  times. 

Pyogenic  bacteria  are  most  frequently  introduced  into  the 
body  through  the  agency  of  injuries  and  wounds  of  various 
sorts.  They  are  very  widely  disseminated  in  nature  and  have 
been  found  clinging  to  various  objects,  especially  in  city  houses. 
The  infection  of  a  wound  with  pyrogenic  cocci,  when  the  sup- 
puration is  of  a  spreading  character,  such  as  is  most  character- 
istic of  streptococcus  infection,  is  known  in  everyday  language 
as  "blood-poisoning."  It  is  possible  for  infection  to  take  place 
around  hair-follicles  through  the  unbroken  skin.  In  such  in- 
stances the  suppurative  inflammation  first  shows  istelf  in  a 


PATHOGENIC    BACTERIA.  287 

minute  red  pimple  with  a  hair  in  the  center.  The  pimple  pres- 
ently becomes  a  pustule.  The  process  may  cease  at  this  point, 
or  it  may  be  only  the  commencement  of  a  large  carbuncle  with 
a  central  slough.  Such  infection  has  been  produced  experi- 
mentally on  the  human  skin  by  rubbing  in  cultures  of  Staphy- 
lococcus  pyogenes  aureus.  It  is,  furthermore,  the  constant 
experience  of  post-mortem  examiners  that  infection  may  occur 
around  the  hair-follicles  when  no  wound  of  the  skin  has  been 
inflicted. 

In  many  instances,  infection  with  the  pyogenic  bacteria 
follows  upon  some  preexisting  infection;  this  happens,  for  in- 
stance, in  tuberculosis,  when  tuberculous  lungs  become  in 
fected  with  Streptococcus  pyogenes,  leading  to  the  formation 
of  a  cavity.  Secondary  infection  with  pyogenic  bacteria  is 
frequently  due  to  the  Streptococcus  pyogenes,  often  also  to  the 
Micrococcus  lanceolatus. 

Sometimes  it  is  impossible  to  detect  the  point  of  entrance  of 
pyogenic  organisms.  In  view  of  the  observation  that  tubercle 
bacilli  pass  through  the  uninjured  mucosa  without  leaving 
any  local  lesion,  but  setting  up  the  disease  in  places  remote 
from  the  point  of  entrance,  it  may  be  surmised  that  the  pyo- 
genic organisms  may  enter  the  body  without  leaving  any  crace 
of  their  point  of  entry. 

The  severe  general  symptoms,  familiar  to  every  physician, 
often  accompanying  acute  suppuration,  indicate  the  formation 
of  toxic  bacterial  products  and  their  absorption.  Experimen- 
tal evidence  of  the  formation  of  such  toxic  products  is  not  so 
clear,  however,  for  the  pyogenic  organisms  as  for  some  of  the 
other  bacteria.  It  has  been  shown  that  cultures  of  Staphy- 
lococcus  pyogenes  aureus,  in  which  the  bacteria  have  been 
killed,  are  capable  of  producing  suppuration  in  the  lower 
animals. 

The  pyogenic  bacteria  play  a  somewhat  different  part  in 
producing  disease,  which  is  fully  as  important  as  the  typical 


288  MANUAL   OF    BACTERIOLOGY. 

suppuration  seen  in  an  abscess.  This  happens  when  the  sup- 
purative  condition  is  complicated  by  other  pathological  proc- 
esses, or  when  there  is  inflammation  of  another  variety  with- 
out suppuration.  These  differences  in  their  action  depend 
largely  upon  the  organ  affected.  One  such  condition  is  osteo- 
myelitis, which  is  suppuration  occurring  in  bone,  but  which 
does  not  present  the  ordinary  picture  of  pus-formation  owing  to 
the  hard  and  unyielding  character  of  the  tissue.  Other  condi- 
tions of  very  great  importance  are  meningitis,  pericarditis, 
pleuritis,  croupous  and  broncho-pneumonia,  peritonitis  and 
endocarditis.  It 'will  be  observed  that  these  affections  are,  for 
the  most  part,  inflammations  of  the  serous  membranes.  Such 
inflammations,  when  they  are  produced  by  pyogenic  bacteria, 
are  likely  to  be  of  great  severity,  accompanied  by  the  formation 
of  fibrinous  exudates;  pus-formation  may  or  may  not  be  pres- 
ent. We  find  that  the  cause  at  times  is  the  Staphylococcus 
pyogenes  aureus;  this  is  often  the  case  in  malignant  endocar- 
ditis. Generally  speaking,  in  such  inflammations  the  Strep- 
tococcus pyogenes,  the  Staphylococcus  pyogenes  aureus  and  the 
pneumococcus  occur  most  commonly,  although  they  are  by  no 
means  the  only  organisms  found.  Many  cases  of  peritonitis 
show  the  presence  of  B.  coli  communis,  either  in  combination 
with  other  bacteria  or  alone.*  This  is  explained  by  the 
proximity  of  the  intestine,  and  especially  by  the  frequent  oc- 
currence of  peritonitis  after  perforation  of  the  intestine.! 

The  process  of  absorption  as  it  occurs  in  the  peritoneum,  as 
shown  by  Buxton,  and  TorreyJ  is  as  follows: 

Solid  particles,  bacteria  or  other  particles,  if  injected  into  the 
peritoneal  cavity  are  quickly  taken  up  into  the  lymph  channels 
of  the  diaphragm  thence  they  are  transported  through  the 
lymph-glands  into  the  thoracic  duct  and  the  blood-vessels.  At 

*Flexner.     Etiology,  etc.,  of  Peritonitis.     Philadelphia   Medical  Journal. 
November  12,  1898. 
t  Buxton  and  Torrey. 
iJourn.  Med.  Res.     XV.,  1906.     pp.  1-88.     Ibid.  XVI.,  1907. 


PATHOGENIC    BACTERIA.  289 

first  the  particles  lie  free  in  the  various  organs  into  which  they 
are  carried,  but  soon  they  are  taken  up  by  phagocytes.  The 
makrophages  are  the  elements  mainly  concerned  in  the  process. 
On  the  injection  of  typhoid  bacilli  into  the  peritoneum  of 
rabbits  there  occurs  an  explosive  destruction  of  many  of  the 
bacilli,  but  those  which  are  not  so  destroyed  are  taken  up  as 
described  above. 

In  inflammations  of  mucous  membranes  the  common  pyo- 

-    *  ,   . 


FIG.  64. — Staphylococcus  pyogenes  aureus  in  pus  stained  by  Gram's 
method.     (X  1000.) 

genie  organisms  are  frequently  the  cause,  though  other  organ- 
isms are  occasionally  responsible.  In  acute  bronchitis,  pneu- 
mococci  and  streptococci  were  found  by  Ritchie*  to  be  the 
commonest  causes. 

In  inflammations  of  the  middle  ear  the  principal  causes  are 
the  pneumococcus,  the  streptococcus  and  the  Staphylococcus 
aureus  and  albus.f 

*  Ritchie.  Journal  Pathology  and  Bacteriology.  Vol.  VII.'  December, 
1900. 

fHasslauer.  CentralUatt  fur  Bakteriologie.  XXXII.  Ref.  1902,  p.  174. 
Compare  ibid.,  pp.  240  and  246. 

19 


290 


MANUAL   OF   BACTERIOLOGY. 


In  25  cases  of  acute  cystitis  in  women  Brown*  found  B.  coli 
communis,  15  times;  S.  pyogenes  albus,  5  times;  S.  pyogenes 
aureus,  twice;  B.  typhosus,  once;  B.  pyocyaneus,  once;  B. 
proteus  vulgaris,  once. 

A  number  of  investigators  have  recovered  organisms  re- 
sembling the  pyogenic  cocci  from  cases  of  acute  articular 
rheumatism.  Most  frequently  a  diplococcus  or  short  strepto- 
coccus has  been  found,  which  has  sometimes  produced  ar- 
thritis and  endocarditis  when  inoculated  into  rabbits.  But 
Cole'st  investigations  seem  rather  unfavorable  to  the  view 
that  this  is  the  specific  organism  for  acute  articular  rheumatism. 
On  the  other  hand  Beattie  J  comes  to  the  conclusion  that  the 
organism  is  specific  for  the  disease,  and  that  M.  rheumaticus, 
is  not  an  attenuated  streptococcus. 

From  a  point  where  there  is  suppuration  or  other  localized 
infection,  pyogenic  bacteria  may  enter  the  circulation  and  be- 
come widely  disseminated  throughout  the  body.  That  hap- 
pens very  commonly  in  malignant  endocarditis.  In  this  man- 
ner secondary  or  metastatic  abscesses  may  be  produced  in  the 
most  diverse  organs. 

In  making  a  diagnosis  in  such  cases,  Rotch  and  Low§  recom- 
mend taking  not  less  than  5  c.c.  of  blood.  A  large  antitoxin 
syringe  serves  the  purpose.  This  is  inserted  into  the  most 
prominent  vein  at  the  elbow  joint,  after  scrubbing  with  soap 
and  water,  and  washing  with  ether  and  alcohol,  and  a  corrosive 
sublimate  pad  tied  above.  Negative  results  they  believe  to 
be  of  value  in  prognosis.  They  believe  the  method  offers 
valuable  means  of  diagnosis  in  obscure  conditions,  cryptogenic 
septicemia  and  autointoxication. 


*  Johns  Hopkins  Hospital  Reports.     Vol.  X.     1902. 
f  Cole.     Experimental  Streptococci! 


:cus  Arthritis  in  Relation  to  the  Etiology  of 

Acute  Articular  Rheumatism.  Journal  of  Injections  Diseases.  Vol.  I.  No.  4. 
Nov.  5,  1904.  P.  714. 

\Journ.  Exper.  Med.     Vol.  IX.,  1907.     pp.  186-206. 

§Journ.Am.Med.  Assn.     Vol.  XL VIII.,  No.  3.    Jan.  19,  1907.  pp.  186-189. 


PATHOGENIC    BACTERIA. 


291 


The  term  pyemia  is  used  to  describe  the  dissemination  of 
pyogenic  bacteria  in  the  circulating  blood,  with  the  formation 
of  metastatic  abscesses. 

Staphylococcus  Pyogenes  Aureus. — A  micrococcus  of 
variable  size,  arranged  in  irregular  clumps,  sometimes  in  pairs; 
about  0.8  to  0.9  /*  in  diameter;  not  motile  (Fig.  65).  It  stains 
by  Gram's  method;  it  is  a  facultative  anaerobe;  grows  rapidly, 
best  at  30°  to  37°  C.  It  liquefies  gelatin.  Upon  gelatin  plates 


FIG.  65. — Staphylococcus  pyogenes  aureus,  pure  culture.     (X  1000.) 


small  colonies  appear  at  the  end  of  about  two  days.  It  grows 
well  upon  all  the  culture-media.  Milk  is  coagulated.  It  does 
not  lead  to  fermentation  with  the  production  of  gas,  but  pro- 
duces various  acids. 

The  growths  in  the  first  place  are  pale,  subsequently  be- 
coming golden-yellow  in  color,  but  only  in  the  presence  of 
oxygen.  This  color  appears  well  on  all  media,  and  is  especially 
distinct  on  potato.  Sometimes  the  color  is  slow  in  developing. 

The  variability  of  resistance  shown  by  the  organism  to  higher 


292  MANUAL    OF    BACTERIOLOGY. 

temperatures  was  shown  by  Liibert*  was  shown  to  depend 
upon  the  manner  of  application.  Suspensions  in  water  were 
killed  in  30  minutes  at  50°  C.  On  dried  threads  it  resisted  60°  C. 
for  one  hour.  Aside  from  the  variation  shown  by  different  strains 
of  the  staphylococcus,  the  medium  in  which  it  is  exposed  to 
heat  has  decided  influence  upon  the  effect  of  the  heat,  the  num- 
ber of  cocci  used  in  the  test  also  has  a  decided  influence.  But 
one-half  to  one  hour  at  80°  C.  appears  to  kill  the  organism 
under  all  conditions.  Cold  appears  to  have  no  effect.  Very 
variable  results  have  been  observed  by  different  authorities  on 
the  effect  of  different  chemical  agents  as  well  as  of  electricity  and 
the  Rontgen  rays.  Corrosive  sublimate  in  the  strength  of  2-1000 
requires  two  or  three  hours.  The  many  statements  in  regard  to 
the  use  of  germicides  in  this  connection  are  confusing.  In  prac- 
tice everything  contaminated  with  pus  containing  the  organism 
should  be  burned  or  sterilized  in  the  autoclave  for  safety.  It  is 
not  killed  by  drying  alone.  In  the  same  specimen  the  micro- 
cocci  may  have  quite  different  resisting  powers  to  chemical 
germicides.  Some  of  the  individual  cells  are  destroyed  by 
i- 1 ooo  solution  of  bichloride  of  mercury  in  five  minutes;  others 
survive  exposure  to  this  solution  for  from  ten  to  thirty  minutes. 

Sterilized  cultures  introduced  into  animals  may  produce 
local  suppuration.  The  cells  contain  intracellular  toxic  sub- 
stances.f 

As  has  already  been  mentioned,  the  Staphylococcus  pyo- 
genes  aureus  is  the  commonest  of  the  pyogenic  bacteria  in  man. 
It  has  been  obtained  from  a  great  variety  of  sources,  and  ap- 
pears to  be  able  to  exist  as  a  saprophyte.  It  has  been  found 
on  the  skin,  in  the  mouth,  in  the  nasal  and  pharyngeal  mucus, 
and  also  in  the  alimentary  canal.  It  has  furthermore  been  de- 
tected in  the  air  and  in  dust.  It  appears  to  find  the  conditions 
necessary  for  its  existence  in  the  vicinity  of  human  habitations. 

*Cited  in  Kolle  and  Wassermann.     Vol.  III.,  1903.  p.  112. 
fMorse.     Journal.  Experimental  Medicine.     Vol.  I.,  p.  263. 


PATHOGENIC    BACTERIA. 


293 


Culture  of  the  Staphylococcus  pyogenes  aureus  vary  con- 
siderably in  virulence.  These  variations  are  sometimes  to  be 
explained  through  cultivation  on  unfavorable  media  or  repeated 
transplantation  from  one  medium  to  another;  but  at  times  the 
diminished  virulence  is  due  to  unknown  causes.  The  lower 
animals  used  for  experiments  are  not  as 
readily  infected  as  man.  The  local  intro- 
duction in  rabbits  or  guinea-pigs  of  a  part 
of  a  culture  of  Staphylococcus  pyogenes 
aureus  may  be  entirely  without  effect.  The 
use  of  a  very  large  dose,  or  the  addition  at 
the  same  time  of  some  kind  of  irritant,  may 
produce  an  abscess.  Large  amounts  of 
cultures  in  bouillon  may  often  be  injected 
into  the  peritoneal  cavity  of  the  dog  without 
effect,  when  the  simultaneous  addition  of  a 
piece  of  sterile  potato  or  an  injury  to  the 
gut  may  lead  to  fatal  peritonitis.  Intro- 
duction of  fluid  cultures  into  the  venous 
circulation  of  the  rabbit  generally  produces 
metastatic  abscesses  in  the  kidneys,  the 
heart- muscle  and  the  voluntary  muscles, 
and  causes  death. 

In  man  this  organism  frequently  produces 
suppuration  of  a  merely  localized  character, 
such  as  we  are  familiar  with  in  boils  and 
carbuncles,  but  it  may  also  cause  gener- 
alized infection.  It  has  been  shown  to  be 
the  usual  cause  of  infectious  osteomyelitis. 
Osteomyelitis  has  been  produced  experimentally  in  rabbits 
by  the  injection  of  the  Staphylococcus  pyogenes  aureus,  both 
with  and  without  previous  injury  to  the  bone  which  becomes 
affected.  Ulcerative  endocarditis  has  on  numerous  occasions 
been  shown  to  be  due  to  this  organism.  It  has  been  found 


FIG.  66. — Staphy lo- 
co ecus  pyogenes 
aureus.  Gelatin  cul- 
ture, one  week  old. 


294  MANUAL    OF    BACTERIOLOGY. 

possible  to  produce  ulcerative  endocarditis  experimentally  in 
animals  by  the  injection  of  the  Staphylococcus  pyogenes  aureus 
when  the  valves  of  the  heart  have  first  been  mechanically  in- 
jured. The  Staphylococcus  pyogenes  aureus  has  also  been 
found  in  acute  abscesses  of  the  lymph-nodes,  tonsils,  parotid 
gland  and  mammary  gland,  in  suppurating  joint  affections 
and  empyema.  It  appears,  furthermore,  in  acute  inflam- 
mation of  the  serous  membranes — pleuritis,  pericarditis, 
peritonitis — although  less  frequently  than  the  Streptococcus 
pyogenes. 

Staphylococcus  Pyogenes  Albus. — In  form  and  manner 
of  growth  this  organism  behaves  like  the  Staphylococcus  py- 
ogenes aureus,  with  the  exception  that  it  produces  no  colored 
growths  and  its  cultures  appear  white.  Its  pathogenic  prop- 
erties are  less  marked,  and  it  is  a  less  frequent  cause  of  sup- 
puration than  the  Staphylococcus  pyogenes  aureus.  It  has, 
however,  been  found  in  acute  abscesses  on  numerous  occasions. 

Staphylococcus  Epidermidis  Albus. — According  to  Welch, 
the  epidermis  of  man  contains  with  great  regularity  the  organ- 
ism to  which  he  gave  the  above  name,  and  which  he  considers 
to  be  a  variety  of  Staphylococcus  pyogenes  albus.  It  grows 
on  all  ordinary  media,  it  liquefies  gelatin,  and  coagulates  milk 
more  slowly  than  the  ordinary  Staphylococcus  pyogenes  albus. 
It  is,  furthermore,  possessed  of  less  marked  pus-producing - 
tendencies.  Welch  found  it  impossible  to  sterilize  the  skin 
so  as  to  remove  this  micrococcus  from  it.  The  organism  is 
usually  innocuous.  It  has  been  found  in  healthy  wounds  on 
numerous  occasions.  It  is  capable  of  causing  trouble  in 
wounds  when  necrotic  or  strangulated  tissues  are  present,  or 
where  a  foreign  body  like  a  drainage-tube  has  been  left  in  the 
wound.  It  is  a  common  cause  of  stitch  abscesses. 

Streptococcus  Pyogenes. — Appears  as  micrococci  ar- 
ranged in  chains,  usually  in  pairs,  when  the  adjacent  cocci 
may  be  flattened.  Sometimes  the  chains  are  very  long.  The 


PATHOGENIC    BACTERIA.  295 

diameters  of  the  cocci  vary  from  0.4  to  i  /*.  Attempts  have 
been  made  to  create  varieties  of  streptococci  according  to  the 
length  of  the  chains.  On  that  basis  a  Streptococcus  brevis  and 
a  Streptococcus  longus  have  been  described. 

Buerger*  makes  a  tentative  division  of. streptococci  into  those 
which  (i)  ferment  dextorse,  levulose,  galactose,  maltose, 
saccharose,  lactose,  inulin,  dextrin  and  mannite;  (2)  those  which 
ferment  all  but  mannite;  (3)  those  which  ferment  all  but 
inulin  (4)  all  but  inulin  and  mannite;  (5)  all  but  inulin  and 
lactose;  (6)  all  but  inulin,  mannite,  and  saccharose. 

The  Streptococcus  pyogenes  is  not  motile.  It  stains  by 
Gram's  method.  Escherichf  recommended  for  the  staining 
of  the  streptococci  found  in  the  intestinal  canal  the  following 
modification  of  the  Gram-Weigert  procedure.  It  is  probably 
available  for  streptococci  in  general. 

THE  STAIN. 

A.  Gentian  violet,  5  gms. 
Water,  200  c.c. 
Boil  a  half  hour  and  filter. 

B.  Absolute  alcohol,  n  c.c. 
Anilin  oil,  3  c.c. 

Add  3  parts  of  A  to  i  part  of  B.     This  stain  keeps  for  about  2  or  3  weeks. 

THE  DISCHARGING  FLUID. 

Iodine-iodide  of  potassium  solution  (i  part  iodine,  2  parts  iodide  of  potas- 
sium, 60  parts  water),  and  analine  xylol  mixed  in  equal  parts.  Moreover, 
pure  xylol.  For  contrast  stain  saturated  alcoholic  solution  of  fuchsin  diluted 
with  equal  parts  of  absolute  alcohol. 

By  the  method  of  Hiss  (page  49)  capsules  may  sometimes  be 
demonstrated.  It  is  facultatively  anaerobic;  grows  best  in  the 
incubator;  more  slowly  at  room  temperature,  and  does  not 
liquefy  gelatin.  In  gelatin  plates  it  produces  small,  round, 
white,  punctiform  colonies  which  are  slow  of  development,  and 

*  Journal.  Exper.  Med.     Vol.  IX.,  1907.     pp.  428-435. 

t  V.  Lingelsheim  in  Kolle  and  Wassermann.     Vol.  III.,  1903.     p.  337. 


296  MANUAL    OF    BACTERIOLOGY. 

are  visible  only  after  about  two  or  three  days.  It  .grows  on  the 
ordinary  media,  but  according  to  some  authors  it  does  not  grow 
on  potato.  Milk  may  or  may  not  be  coagulated.  The  growths 
are  never  very  luxuriant,  and  may  die  out  after  a  few  trans- 
plantations. 

Streptococcus  pyogenes  longus  is  not  killed  with  certainty 
when  suspended  in  normal  salt  solution  and  heated  at  60°  C. 
for  one  hour.*  The  Streptococcus  pyogenes  occurs  frequently 


C 


n 


FIG.  67. — Streptococcus  pyogenes,  from  a  pure  culture.     (X  1000.) 

on  the  mucous  surfaces  of  the  healthy  body.  It  is  often  found 
in  pus,  especially  pus  of  spreading  inflammations  of  the  kind 
known  as  cellulitis.  This  organism  is  the  commonest  infectious 
agent  in  puerperal  fever,  metritis  and  peritonitis.  It  occurs 
commonly  in  inflammations  of  the  serous  membranes — pleu- 
ritis,  pericarditis  and  peritonitis.  It  has  been  discovered  many 
times  in  ulcerative  endocarditis  and  in  bronchopneumonia.  It 
is  frequently  present  in  the  false  membrane  found  in  genuine 

*V.  Lingelsheim  in  Kolle  and  Wassermann.     Ed.  III.,  1903.     p.  318. 


PATHOGENIC    BACTERIA.  297 

diphtheria.  It  is  also  the  cause  of  many  of  the  pseudomem- 
branous  or  so-called  "diphtheritic"  affections  of  the  throat 
where  the  Klebs-Loffler  bacillus  of  diphtheria  is  wanting. 
These  cases  may  be  indistinguishable  clinically  from  genuine 
•diphtheria,  and  their  nature  can  be  revealed  only  by  bacterio- 
logical examination.  They  are,  however,  as  a  rule,  milder  than 
genuine  diphtheria.  The  pseudomembranous  affections  of 
the  throat  which  occur  in  scarlet  fever  and  measles  are 


FIG.  68. — Streptococcus  pyogenes  in  pus,  Gram's  stain.     (X  1000.) 

generally  caused  by  the  Streptococcus  pyogenes,  although  those 
diseases  may  be  complicated  by  genuine  diphtheria.  Strep- 
tococci are  very  commonly  present  in  the  throat  in  scarlet 
fever,*  and  sometimes  occur  in  the  blood.  Ruedigerf  using 
blood  agar  found  streptococcus  present  in  the  throat  in  all  the 
cases  of  scarlatina — 75  cases — which  he  examined.  HektoenJ 
points  out  that  while  it  is  not  yet  established  beyond  question 

*Weaver.     American  Medicine.     April  18.  1903. 
\Journ.  Injections  Diseases.     Vol.  III.,  1906.  pp.  755-771- 
\Journ.  Am.  Med.  Assn.     Vol.  XL VIII  ,  No.  14,  April  6,  1907.    pp.  1158- 
ii  60. 


298 


MANUAL    OP    BACTERIOLOGY. 


that  the  streptococcus  is  the  cause  of  scarlet  fever,  and  that 

it  seems  more  likely  that  this  organism  is  a  secondary  invader, 

nevertheless  the  Streptococcus  pyo- 
genes  is  always  present  in  large 
numbers  in  the  throats  of  affected 
individuals,  and  causes  many  serious 
complications  or  it  may  be  a  fatal 
termination.  Some  observers  have 
produced  evidence  going  to  show 
that  scarlet  fever  is  caused  by  strep- 
tococci. Streptococci  are  very  often 
found  in  the  pustules  of  small-pox, 
and  may  also  appear  in  the  blood. 

The  Streptococcus  pyogenes  is 
pathogenic  for  mice  and  rabbits,  but 
the  virulence  is  very  variable.  This 
may  sometimes  be  increased  by 
passing  through  a  number  of  ani- 
mals in  succession,  but  is  rapidly 
lost  in  artificial  cultures.  It  is  said 
that  the  virulence  is  best  maintained 
when  cultures  on  gelatin,  after  forty- 
eight  hours'  growth,  are  kept  in  a 
cool  place,  as  in  the  ice-chest. 

A  serum  of  uncertain  value  derived 
from  an  immunized  horse  or  ass  has 
been  prepared  by  Marmorek  for  the 
cure  of  streptococcus  infection. 

A  number  of  other  sera  have  been 
prepared  for  combating  streptococ- 
cus infection.  These  have  been 

used  in  cases  of  streptococcus  infection  in  human  beings;  also 

in  cases  of  scarlet  fever. 

Van  de  Velde,  and  others  have  prepared  a  so-called  "polly- 


FIG.  69. — Streptococcus  pyo- 
genes, culture  on  agar  (slightly 
enlarged). 


PATHOGENIC    BACTERIA. 


299 


valent"  streptococcus  serum  by  using  streptococci  from  various 
sources  for  the  injection  of  animals.  The  blood-serum  of  the 
injected  animals  constitute  the  pollyvalent  serum.  It  is  im- 
possible at  present  to  speak  definitely  in  regard  to  the  value  of 
antistreptococcus  serum  since  some  seem  to  have  obtained  very 
favorable  results  with  it  in  practice,  while  others  report  no 
observable  improvement  in  cases  treated  with  it.  Since  the 
streptococci  belong  to  that  class  of  bacteria  which  form  endo- 


FIG.  70. — Micrococcus  tetragenus  in  pus  from  a  large  abscess  on  the  arm, 
showing  capsule,  Gram's  stain  and  eosin.     (X  1000.) 

toxins;  the  problem  of  producing  an  antitoxin  is  an  unsatisfac- 
tory matter,  to  say  the  least. 

It  is  said  that  streptococci  may  be  agglutinated  by  serum 
from  animals  immunized  with  streptococcus. 

Coley*  recommends  the  use  of  mixed  culture  of  Streptococ- 
cus pyogenes  (or  S.  erysipelatos)  and  B.  prodigiosus  in  the 
treatment  of  inoperable  sarcomata.  The  cultures  with  which 
he  was  very  successful  in  a  number  of  cases  were  prepared  for 

*Reprint  from  Med.  Record.     July  27,  1907. 


300  MANUAL   OF    BACTERIOLOGY. 

him  by  Buxton.  The  streptococcus  was  grown  in  beef-broth 
for  ten  days  and  the  culture  then  inoculated  with  B.  prodigiosus 
and  the  mixed  culture  grown  for  another  ten  days.  These  cul- 
tures, sterilized  at  60°  C.,  were  injected  into  the  tumors  without 
filtering. 

Streptococcus  of  Erysipelas. — The  cause  of  erysipelas  is 
a  streptococcus  which  in  all  essential  respects — in  its  mor- 
phology, its  growth  on  culture- media,  its  behavior  with  stains 
and  its  pathogenic  properties— corresponds  to  the  Streptococcus 
pyogenes.  It  is  probable  that  these  organisms  are  identical, 
though  the  clinical  manifestations  in  erysipelas  are  sufficiently 
characteristic  to  justify  the  clinician  in  making  a  distinction 
between  this  on  the  one  hand,  and  the  other  manifestations  of 
streptococcus  infection  on  the  other.  In  erysipelas,  conta- 
giousness is  a  most  marked  feature. 

Micrococcus  Tetragenus.— Found  in  the  cavities  in  the 
lungs  of  pulmonary  tuberculosis,  in  sputum  and  in  pus.  The 
micrococci  are  enclosed  in  a  transparent  capsule,  best  seen  in 
preparations  from  the  tissues  of  inoculated  animals,  and  are 
arranged  in  pairs  or  in  fours;  about  i  ^  in  diameter;  not  motile; 
stain  by  Gram's  method.  It  grows  well  at  the  room  tempera- 
ture, but  rather  slowly;  is  a  facultative  anaerobe;  does  not 
liquefy  gelatin.  Gelatin  plates  show  little,  white,  punctiform 
colonies,  which,  with  the  low  power,  are  finely  granular,  and 
have  a  peculiar  glassy  shimmer;  in  stab-cultures  the  growths 
appear  as  little  colonies  along  the  line  of  puncture.  On  agar, 
round  white  colonies  form,  which  do  not  tend  to  spread.  It 
produces  a  thick,  slimy  film  on  potato  and  a  broad,  white, 
moist  growth  on  blood-serum.  This  organism  is  only  oc- 
casionally found  in  pus.  It  is  pathogenic  for  white  and  gray 
mice  and  guinea-pigs,  jbut  not  for  rabbits.  It  may  produce 
septicemia  or  only  a  localized  suppuration  in  guinea-pigs.  In 
white  mice  general  septicemia  results  on  inoculation  and  the 
Micrococcus  tetragenus  is  found  in  the  blood  and  in  the  great 


PATHOGENIC    BACTERIA.  30! 

viscera.  White  mice  usually  die  in  from  two  to  six  days; 
guinea-pigs  in  from  four  to  eight  days  from  inoculations. 

Micrococcus  Lanceolatus  (Micrococcus  pneumonias  crou- 
posae;  Micrococcus  Pasteuri;  Diplococcus  pneumonias;  Micro- 
coccus  of  Sputum  Septicemia;  Streptococcus  lanceolatus  Pas- 
teuri; and  Pneumococcus  of  Frank  el). — This  organism  was 
discovered  by  Sternberg  in  his  saliva  in  1880,  and  -afterward 
demonstrated  to  be  the  cause  of  lobar  pneumonia  by  Frankel 
and  Weichselbaum.  The  micrococci  usually  occur  in  pairs. 
The  pair  of  micrococci,  in  its  most  typical  form,  appears  like  a 
couple  of  curved  triangles  with  their  bases  close  to  each  other. 
The  outline  is  usually  described  as  being  lancet-shaped.  The 
micrococci  are  frequently  oval  or  round;  they  often  form  chains. 
In  preparations  made  from  the  blood  of  infected  animals  or 
from  pneumonic  sputum  each  pair  of  micrococci  is  often  seen 
in  stained  preparations  to  be  surrounded  by  a  capsule;  though 
even  in  such  preparations  the  capsule  is  sometimes  difficult 
to  demonstrate;  the  capsule  is  not  usually  seen  in  preparations 
made  from  cultures.  For  methods  of  demonstrating  the 
capsule  see  pages  49  and  121.  The  penumococcus  is  not  motile. 
It  £tains  by  Gram's  method.  It  grows  on  potato,  but  the 
growth  is  not  visible  to  the  naked  eye* 

As  with  most  other  pathogenic  bacteria,  the  indentification 
of  the  pneumococcus  is  uncertain  from  observation  of  its 
morphology  and  staining  properties  alone.  These  have  to  be 
supplemented  as  in  other  cases  by  cultures  and  animal 
experiments. 

It  is  facultatively  anaerobic.  It  grows  only  at  elevated 
temperatures,  preferably  about  35°  to  37°  C.  Gelatin  is  not 
liquefied.  It  grows  well  upon  agar,  upon  blood-serum  and 
upon  Guarnieri's  medium  (page  75).  Milk  usually  becomes 
acid,  and  may  or  may  not  be  coagulated.  The  colonies  are  seen 
in  their  characteristic  form  upon  agar,  and  are  developed  after 

*  Weichselbaum  in  Kolle  and  Wassermann.     Bd.  III.,  1903.  p.  201. 


302  MANUAL   OF    BACTERIOLOGY. 

about  forty-eight  hours,  appearing  as  minute,  whitish,  trans- 
lucent, circular  growths. 

As  a  means  of  differentiation  between  the  pneumococcus  on  the  one  hand 
and  the  streptococcus  on  the  other,  Hiss*  devised  a  medium  consisting  of 
nutrient  agar  to  which  inulin  is  added.  Hiss  showed  that  the  pneumococcus 
ferments  this  substance  in  sugar  free  media,  while  streptococcus  does  not  fer- 
ment it.  In  fact  there  is  a  group  of  organisms  morphologically  and  culturally 
like  the  pneumococcus  which  do  not  ferment  indulin.  Ruedigerf  has  made 
use  of  Hiss  inulin  agar  for  the  isolation  of  the  pneumococcus,  and  he  gives  the 
following  directions  for  its  preparation  and  use : 

(a)  Peptone  (Witte)  10  c.c. 

Agar-agar  15  c.c. 

Sugar-free  broth  (neutral)  1,000  c.c. 

Dissolve  by  boiling  for  one  hour,  adding  water  from  time  to  time  to  com- 
pensate for  evaporation.  Heat  in  the  autoclave  for  15  or  20  minutes,  clarify 
with  egg,  filter  through  cotton  and  make  up  to  800  c.c.  with  distilled  water. 

(b)  Dissolve  15  grams  of  pure  inulin  in  200  c.c.  of  distilled  water,  mix  this 
solution  with  (a),  add  20  of  a  5  per  cent,  solution  of  litmus  (Merk's  highest 
purity),  tube  and  sterilize  in  the  autoclave  under  10  pounds'  pressure  for  15 
minutes.  Some  stains  of  pneumococcus  will  not  grow  in  this  medium;  it  may 
be  improved  by  adding  i  c.c.  of  ascetic  fluid  to  each  tube  before  use.  In 
this  medium  the  pneumococcus  in  24  to  96  hours  at  37°  C.  show  red  colonies 
agiinst  a  blue  background. 

Berry  J  concludes  from  her  investigations  that  the  pneumo- 
coccus undergoes  decided  changes  under  artificial  cultivation, 
among  others  in  its  loss  of  power  of  fermenting  inulin.  These 
changes  may  be  permanent  or  they  may  return  under  favorable 
conditions.  The  change  in  morphology  is  seen  in  a  tendency 
toward  a  streptococcus  type.  Loss  of  virulence  is  always 
lost  on  artificial  media,  but  is  enhanced  by  passage  through 
animals.  Berry  concludes  in  regard  to  the  inulin  reaction  that 
too  much  reliance  should  not  be  placed  upon  it,  and  that  a 
negative  result  is  not  sufficient  to  exclude  the  pneumococcus, 
particularly  where  the  organism  tested  has  been  cultivated  for 
a  long  time  on  artificial  culture  media. 

*Journ.  Experimental  Medicine.     1905,  No.  7,  p.  317. 
•\Journ.  Infectious  Diseases.     Vol.  III.,  1906,  p.  317. 
JBerry.     Journ.  Infect.  Dis.     Vol  IV.,  1907,  pp.  93-107. 


PATHOGENIC    BACTERIA.  303 

Extensive  studies  of  the  pneumococcus  were  made  under  the  auspices  of  the 
Medical  Commission  for  the  Investigation  of  Acute  Respiratory  Diseases  of 
the  Department  of  Health  of  the  City  of  New  York.*  These  studies  covered 
the  matter  of  the  occurrence  of  the  organisms  in  the  throats  of  healthy  in- 
dividuals, from  persons  suffering  from  various  diseases;  the  various  cultural 
peculiarities,  the  comparison  between  the  pneumococcus  and  streptococci,  the 
agglutination  reaction,  staining  peculiarities,  and  pathogenic  properties. 

Schottmiillert  and  Rosenowf  independently  of  one  another 
found  that  the  pneumococcus  presented  very  characteristic  ap- 
pearance when  grown  on  agar  to  which  rabbit  or  human  blood 
is  added.  The  medium  recommended  by  Rosenow  is  pre- 
pared by  adding  0.3  to  0.5  c.c.  of  sterile  defibrinated  blood  to 
the  tube  of  melted  agar  cooled  to  45°  C.  Upon  this  medium 
the  colonies  grow  larger  than  on  other  media,  and  they  have  a 
distinct  green  color,  and  are,  furthermore,  surrounded  by  a  nar- 
row zone  in  which  the  blood-corpuscles  are  destroyed.  This 
zone  is  always  opaque  and  has  a  greenish  tinge.  The  pneumo- 
coccus remains  viable  and  retains  its  virulence  for  a  remarkably 
long  time  when  it  is  cultivated  upon  this  medium.  RuedigerJ 
has  shown  that  the  green  color  of  the  colonies  of  pneumococcus 
in  this  medium  is  due  to  the  production  of  an  acid,  probably 
lactic. 

Buerger  and  Ryttenberg§  have  come  to  the  following  con- 
clusions :  The  fermentation  of  inulin  is  of  limited  value  for  the 
differential  diagnosis  between  pneumococci  and  streptococci. 
Pneumococci  may  lose  the  power  to  produce  acid  in  this 
carbohydrate  either  temporarily  or  permanently.  Both  pneu- 
mococci and  streptococci  may  produce  precipitation  in  glucose- 
serum-agar.  Both  may  cause  hemolysis  in  blood-agar.  The 
diagnosis  may  be  made  when  everything  else  fails  by  use  of 
the  capsule  staining  and  animal  inoculation.  An  organism 

*Park  and  Williams;  Collins;  Longcope  and  Fox;  Norris  and  Pappenheimer; 
Duval  and  Lewis;  Buerger;  Hiss  and  others;  Wood;  Longcope.  Journ.  Ex- 
per.  Med.  Vol.  VII.,  1905,  pp.  401-626. 

t Rosenow.     Journ.  Infectious  Diseases.     Vol.  I.,  1904,  pp.  280-312. 

%Journ.  Infect.  Diseases.     Vol.  III.,  1907.     pp.  663-665. 

§  Journ.  Infect.  Diseases.     Vol.  IV.,  1907.     pp.  609-616. 


304  MANUAL   OF    BACTERIOLOGY. 

with  pneumococcus  capsule  staining  and  streptococcus  cultural 
features  may  with  probability  be  diagnosed  as  pneumococcus 
even  though  it  does  not  return  to  the  pneumococcus  type  after 
animal  passage. 

It  is  killed  by  an  exposure  to  a  moist  temperature  of  52°  C. 
for  ten  minutes. 

It  is  best  cultivated  from  the  blood  of  an  animal  which  has 
been  infected  with  the  sputum  of  a  case  of  lobar  pneumonia. 


/ 


J 


FIG.  71. — Pneumococcus  of  Frankel  in  sputum  of  pneumonia,  Gram's 
stain  and  eosin. 

Cultures  need  to  be  transplanted  every  few  days;  they  cannot 
usually  be  propagated  more  than  a  month  or  two  months. 
The  virulence  of  the  organism  for  animals  diminishes  rap- 
idly in  cultures.  It  frequently  grows  as  a  streptococcus  on 
artificial  media.  When  virulent,  it  is  pathogenic  for  mice  and 
rabbits;  less  so  for  guinea-pigs.  In  these  animals  it  is  likely 
to  lead  to  fatal  septicemia  in  twenty-four  to  forty-eight  hours 
when  introduced  subcutaneously  or  into  the  peritoneum  or 
when  liquid  cultures  are  injected  intravenously.  The  blood 


PATHOGENIC  BACTERIA.  30  S 

often  contains  great  numbers  of  the  diplococci.  The  virulence 
of  the  organism  is  very  variable.  In  the  sputum  of  a  case  of 
lobar  pneumonia,  early  in  the  disease,  it  is  likely  to  be  virulent. 
The  virulence  is  best  maintained  by  repeated  inoculations  into 
mice  or  rabbits. 

The  Micrococcus  lanceolatus  has  been  detected  very  fre- 
quently in  the  mouths  of  healthy  individuals.  But  under  these 
conditions  it  is  not,  however,  pathogenic  for  animals  in  many 


FIG.  72. — Pneumoccccus,  showing  capsule,  from  pleuritic  fluid  cf  infected 
rabbit,  stained  by  second  method  of  Hiss. 

instances,  being  found  virulent  in  only  from  15  to  20  per  cent, 
of  such  cases.  While  it  is  unquestionably  the  cause  of  croupous 
or  lobar  pneumonia  in  man  in  most  if  not  in  all  cases,  there 
are  competent  observers  who  hold  that  lobar  pneumonia  is  also 
caused  by  other  bacteria.  In  that  disease  the  characteristic 
lesion  consists  of  an  inflammation  of  the  lung,  involving  large 
areas — usually  one  or  several  lobes.  An  exudate  is  poured  into 
the  air-vesicles,  which  in  the  early  part  of  the  disease  contains 
red  blood-cells,  imparting  the  rusty  color  to  the  sputum.  The 
principal  element  in  the  exudate  is  fibrin;  it  also  contains  leuko- 


306  MANUAL   OF   BACTERIOLOGY. 

cytes.  The  formation  of  fibrin  produces  the  liver-like  consoli- 
dation or  "hepatization."  The  diplococci  can  readily  be 
demonstrated  by  the  Gram  method  in  sections  of  pneumonic 
lung,  which  are  best  stained  by  carmine  and  gentian-violet. 

The  Micrococcus  lanceolatus  can  be  sometimes  detected  in 
large  numbers,  occasionally  almost  unmixed  with  other  bacteria, 
in  the  rusty  sputum  of  lobar  pneumonia,  often  showing  the 
peculiar  unstained  capsule.  On  account  of  its  liability  to  be 
mixed  with  other  forms  of  bacteria  its  presence  in  the  sputum 
of  cases  suspected  of  being  pneumonia  is  not  of  very  great 
value  in  differential  diagnosis,  especially  since  it  is  so  com- 
monly present  in  the  normal  mouth.  In  a  suspicious  case 
its  appearance  in  sputum  in  nearly  pure  culture  may  be 
significant. 

Cultures  from  the  blood  of  cases  of  pneumonia,  where  a  large 
amount  of  blood  is  taken,  have  shown  the  presence  of  the 
pneumococcus  in  a  considerable  proportion  of  the  cases,  es- 
pecially when  severe  or  fatal. 

Rosenow*  found  that  with  proper  technic,  using  large 
quantities  of  blood — 5  to  7  c.c.— the  pneumococcus  can  be  re- 
covered from  the  blood  in  practically  all  cases  of  croupous 
pneumonia,  and  this  method  may  be  employed  with  advantage 
for  diagnosis  in  obscure  cases  of  the  disease.  Wolff  found  that 
the  pneumococcus  is  present  in  the  blood  in  a  large  percentage 
of  cases  even  after  crisis. 

The  Micrococcus  lanceolatus  is  often  also  the  cause  of 
bronchopneumonia  and  of  meningitis.  WallsteinJ  obtained 
it  in  15  out  of  33  cases  of  primary,  and  in  many  cases  of  second- 
ary bronchopneumonia.  It  produces  inflammations  in  other 
situations  as  well,  the  most  important  being  pleuritis,  pericar- 
ditis, endocarditis  and  arthritis.  The  Micrococcus  lanceolatus 

*Journ.  Infectious  Diseases.     Vol.  I.,  1904.     pp.  280-312. 
•\Journ.  Infectious  Diseases.     Vol.  III.,  1906.     pp.  446-451. 
%Journ.  Exper.  Med.     Vol.  VI.,  1905.     pp.  391-400. 


PATHOGENIC    BACTERIA.  307 

may  produce  pseudomembranous  inflammation*  and  also 
ordinary  suppuration,  although  not  very  commonly.  It  seems 
also  capable  of  producing  ordinary  "cold,"  f  acute  catarrh. 

PacchioniJ  found  the  organism  in  pus  from  an  inflamed 
knee-joint  following,  measles  with  bronchopneumonia  and 
pleurisy  as  sequellae.  Furrer  §  also  reports  a  case  of  pneu- 
mococcus  arthritis. 

G.  and  F.  Klemperer  claim  to  have  obtained  toxins  from 
cultures  of  the  pneumococcus,  and  to  have  established  im- 
munity in  animals  with  the  development  in  the  blood  of  anti- 
toxic substances.  Similar  attempts  have  been  made  by  Wash- 
bourn  and  others,  but  the  interpretation  of  their  results  at  the 
present  time  is  not  clear.  The  agglutination  reaction  has  been 
claimed  to  occur  with  the  pneumococcus,  but  it  does  not  yet  ap- 
pear to  have  any  practical  value  in  diagnosis. 

In  regard  to  the  opsonic  index  (page  216)  Wolf  ||  has  this  to 
say :  It  first  decreases,  but  rises  in  favorable  cases,  and  attains 
its  height  soon  after  crisis.  In  unfavorable  cases  it  remains 
persistently  low. 

Organisms  related  to  the  pneumococcus  have  been  described 
under  the  names  of  pseudopneumococcus^f  and  Streptococcus 
mucosus.** 

The  organism  named  by  Rosenbach  Micrococcus  pyo genes 
tenuis  is  probably  only  a  variety  of  the  pneumococcus. 

Micrococcus  Melitensis. — A  micrococcus  found  by  Bruce 
in  cases  of  Malta  or  Mediterranean  fever.  It  is  a  round  or 
slightly  oval  organism,  about  0.5  A*  in  diameter,  occurring 
singly,  in  pairs  or  in  short  chains.  It  is  usually  said  to  be  non- 

*  Gary  and  Lyon.     American  Journal  Medical  Sciences.     Vol.  CXXII. 
fHiss.  Journ.  Expr.  Med.     Vol.  VII.,  1905.     p.  578. 
%Riv.  di  Clin.  Pediatr.     July,  1903. 
§Arch.  for  Pediatrics.     July,  1907. 

\\Journ.  Infectious  Diseases.     Vol.  III.,  1906.     pp.  731-741. 
^[Richardson.     Journal  Boston  Society  of  Medical  Sciences.     Vol.  V.,  1901. 
**  Howard.      Journal   Medical   Research.      Vol.   VI.,   1901.     H.  S.  Journ. 
Exper.  Medicine.     VII.,  1905.     p.  547.     Buerger  eben.     p.  497. 


308  MANUAL   OF    BACTERIOLOGY. 

motile,  though  flagella  have  been  described.  It  is  stained  by 
ordinary  aniline  dyes,  but  not  by  Gram's  method.  It  grows 
slowly,  even  in  the  incubator,  and  more  slowly  at  ordinary 
temperatures.  In  gelatin  the  growth  is  feeble;  there  is  no 
liquefaction.  On  agar  pearly  white  growths  appear  after 
three  or  four  days.  Bouillon  becomes  turbid,  and  later  a 
sediment  is  formed.  On  potato  there  may  be  slight  invisible 
growth. 

A  commission  sent  by  the  Royal  Society  of  England  *  came  to  the  conclusion 
that  Malta  fever  is  spread  through  goats'  milk.  The  report  of  this,  as  it  ap- 
pears in  the  Journal  quoted,  does  not  seem  convincing. 

Malta  fever  occurs  chiefly  about  the  Mediterranean.  It 
has  been  observed  in  India,  in  the  Philippine  Islands  and  in 
Porto  Rico. 

It  is  a  chronic  febrile  disease,  but  not  very  fatal,  accompanied 
by  pains  in  the  joints  and  perspiration.  At  autopsies  the 
organisms  may  best  be  recovered  from  the  enlarged  spleen.. 
Accidental  infection,  sometimes  fatal  in  man,  has  occurred 
from  pure  cultures  on  a  number  of  occasions.  The  disease 
may  be  reproduced  in  monkeys  by  inoculation  with  pure  cul- 
tures. The  agglutination  reaction  is  positive  in  this  disease. 
The  diagnosis  is  best  made  by  applying  this  test  to  the  blood- 
serum  of  the  patient,  with  a  known  pure  culture  of  Micrococcus 
melitensis.t  For  this  purpose  a  suspension  of  an  agar  culture 
is  made  in  normal  salt  solution.  The  diluted  serum  is  added 
so  as  to  secure  a  dilution  of  about  1-50,  but  the  dilutions  used 
have  varied  widely.  Precipitation  quickly  follows  agglutina- 
tion. According  to  Craig,  the  test  may  be  made  on  a  slide, 
examining  with  the  microscope  as  for  the  typhoid  bacillus  (see 
Serum- test  for  Typhoid  Fever). 

*  London  letter  to  Journ.  Am.  Med.  Assn.     June  i,  1907. 

fMusser  and  Sailer.  Philadelphia  Medical  Journal.  December  31,  1898, 
July  8,  1899.  Strong  and  Musgrove.  Ibid.  November  24,  1900.  Curry. 
Journal  Medical  Research.  Vol.  VI.  1901. 


PATHOGENIC    BACTERIA.  309 

Diplococcus  Intracellularis  Meningitidis.*— Found  in 
the  exudate  of  cerebro-spinal  meningitis  by  Weichselbaum; 
isolated  by  Dunham  t  and  others  from  the  upper  air  passages 
in  such  cases. 

Flexner  J  found  that  the  reason  for  the  rapid  death  and  dis- 
integration of  the  organism  which  takes  place  in  24  to  48 
hours  in  the  ice  box  and  less  quickly  in  the  incubator  is  due 
to  an  enzyme  contained  in  the  organism  itself — an  autolytic 
enzyme. 


FIG.  73. — Diplococcus  intracellularis  meningitidis  and  pus-cells.      (X  1000.) 

Flexner  furthermore  found  that  by  using  diluted  serum  of  the 
sheep  instead  of  that  of  human  beings  in  the  agar  medium,  and 
by  the  addition  of  a  calcium  salt  (calcium  carbonate)  to  this 
sheep-serum-agar  medium  a  given  culture  will  remain  alive 
many  weeks. 

Dunham  and  Ward,  assisting  Flexner,  failed  to  find  the  or- 

*  The  writer  is  indebted  for  the  brief  statement  which  it  is  possible  to  give 
here  chiefly  to  the  exhaustive  Report  to  the  Massachusetts  Board  of  Health 
by  Councilman,  Mallory  and  Wright,  1898.  The  photograph  was  made  from 
a  preparation  kindly  furnished  by  Dr.  Mallory.  See  also  Davis  Journ.  Infec. 
Dis.  Vol.  IV.  1907.  pp.  558-681. 

f  Journ.  Inject.  Dis.     Sup.  No.  2.     Feb.,  1906.     pp.  10-20. 

t  Journ.  Exper.  Med.     Vol.  IX.     1907.     pp.  105-141. 


310  MANUAL   OF   BACTERIOLOGY. 

ganism  in  the  throats  and  nasal  cavities  of  a  number  of  domes- 
tic pets  and  of  persons  not  suffering  from  meningitis. 

A  micrococcus  about  the  size  of  the  common  pyogenic  cocci; 
grows  in  fours,  but  more  often  in  pairs  consisting  of  two  hemis- 
pheres separated  by  an  interval  which  does  not  stain;  usually 
found  within  the  pus-cells,  in  which  respect  it  resembles  the  go- 
nococcus.  It  is  stained  by  ordinary  methods  with  the  aniline 
dyes,  and  is  decolorized  by  Gram's  method.  It  does  not  grow  at 
the  room  temperature,  but  only  in  the  incubator;  gelatin  is  not 
available  as  a  culture-medium.  There  is  no  growth  on  potato 
and  scanty  growth  on  agar  or  in  bouillon.  The  development 
is  most  abundant  upon  Loffler's  blood-serum,  when  round, 
white,  shining,  viscid-looking  colonies  with  sharp  outlines  may 
be  seen  in  twenty-four  hours.  The  serum  is  not  liquefied. 
Upon  agar,  or  better  upon  glycerin- agar,  the  colonies  are  flat, 
round,  translucent,  viscid-looking,  having  a  yellowish-brown 
color  under  the  low  power.  The  organism  should  be  trans- 
planted to  fresh  media  frequently,  as  it  rapidly  loses  its  power  of 
reproduction.  Many  of  the  tubes  inoculated  with  the  original 
material  or  with  pure  cultures  show  no  growth. 

It  is  moderately  pathogenic  for  guinea-pigs  and  rabbits 
when  inoculated  into  the  pleura  or  peritoneum.  Meningitis 
and  encephalitis  have  been  produced  in  the  dog  and  goat  by 
inoculation  in  the  meninges. 

Flexner  (loc.  cit.  pp.  141-167)  found  that  injection  of  the 
organisms  into  the  spinal  canal  of  monkeys  produces  a  disease 
the  symptoms  and  lesions  of  which  bear  close  resemblance  to 
those  occurring  in  the  natural  infection  in  man.  Injection 
of  monkeys  into  other  parts  of  the  body  of  monkeys  produce 
only  local  effects. 

This  organism  appears  to  be  the  principal  if  not  the  only 
cause  of  epidemic  cerebro-spinal  meningitis.  The  lesion  con- 
sists of  a  purulent  inflammation  of  the  pia  and  arachnoid,  ex- 
tending into  the  brain-substance,  over  the  cord  and  along  the 


PATHOGENIC   BACTERIA.  31 1 

nerves.  General  invasion  of  the  tissues  of  the  body  seems 
not  to  occur,  but  focal  areas  of  pneumonia  may  be  present. 
Spinal  puncture  in  the  lumbar  region  is  recommended  as  a 
means  of  diagnosis.  The  fluid  should  be  examined  with  the 
microscope  and  by  cultures. 

Flexner  (loc.  cit.  pp.  168-185)  has  made  use  of  antisera, 
which  counteract  the  fatal  effects  of  injections  of  intracellu- 
laris.  He  also  found  that  this  could  be  effected  to  a  less  extent 
by  normal  sera  and  other  fluids.  The  "trials  of  antiserum  ob- 
tained from  the  horse  have  been  tried  in  a  number  of  cases 
upon  human  beings  with  apparently  favorable  results,  and 
Flexner  and  Jobling*  state  that  while  they  are  not  finally  con- 
vinced of  its  value,  they  believe  the  data  so  far  warrants  a 
wider  trial.  The  organism  was  obtained  by  Warfield  and 
Walkerf  from  a  case  of  endocarditis  with  general  septicemia. 

Micrococcus  Gonorrhoeas  (Gonococcus  of  Neisser).— 
Found  in  pus  in  cases  of  gonorrhea.  The  micrococci  generally 
are  in  pairs,  occasionally  in  groups  of  four.  The  cocci  are 
flattened,  the  flattened  sides  facing  each  other,  and  they  are 
often  compared  to  a  pair  of  biscuits.  The  long  diameter  of  the 
pair  of  biscuit-shaped  elements  is  about  1.25  A*.  The  organ- 
isms are  usually  found  attached  to  the  epithelial  cells  or  inside 
of  the  pus-cells;  they  are  also  found  in  smaller  numbers  floating 
free  in  the  fluid.  They  stain  with  ordinary  aniline  dyes,  for 
example,  Loffler's  methylene-blue,  but  not  by  Gram's  method. 

The  fact  (i)  that  the  cocci,  after  the  acute  stage  at  least,  in 
carefully  prepared  specimens  are  always  found  largely  inside 
of  the  pus-cells,  (2)  that  they  are  in  pairs  of  biscuit-shaped 
micrococci,  (3)  that  they  are  not  stained  by  Gram's  method, 
will  serve  to  distinguish  the  gonococcus  from  all  the  other  ordi- 
nary pus-forming  bacteria  and  from  the  meningococcus  and  M. 
catarrhalis.  There  are  other  diplococci  (pseudogonococci), 

*Journ.  Exper.  Med.     Vol.  X.,  1908.  p.  202. 

fBull.  Ayer  Lab.,  Univer.  Penna.  Hosp.     No.  i,  Oct.,  1903. 


3I2 


MANUAL    OF    BACTERIOLOGY. 


probably  non-pathogenic,  which  have  been  found  occasionally 
in  the  vulvo-vaginal  tract  and  in  the  uretha,  which, -it  is  said, 
are  also  decolorized  by  Gram's  method.  Such  organisms  are 
not  likely  to  present  all  the  points  mentioned  as  characteristic 
of  the  gonococcus.  The  recognition  of  the  gonococcus  in  the 
discharges  of  a  case  of  acute  gonorrhea  is  usually  an  easy 
matter.  It  must  be  admitted,  however,  that  in  cases  having 
chronic  discharges,  when  its  detection  is  most  to  be  desired, 


FIG.  74. — Gonococci  and  pus-cells.     (Xiooo.) 

the  diagnosis  may  become  very  difficult  and  is  frequently  im- 
possible, except  by  culture-methods,  owing  to  secondary  in- 
fection with  the  ordinary  pus-forming  or  other  bacteria,  which 
may  be  present  in  larger  numbers  than  the  gonococci 
themselves. 

The  gonococcus  grows  only  in  the  incubator,  and  cannot 
therefore  be  cultivated  upon  gelatin.  Its  cultivation  is,  in  fact, 
a  matter  of  some  difficulty.  The  medium  usually  selected 
is  a  mixture  of  agar  with  human  blood-serum.  The  blood- 


PATHOGENIC    BACTERIA.  313 

serum  from  the  placental  blood  or  pleuritic  or  peritoneal 
transudates,  or  hydrocele  fluid,  has  been  employed.  Human 
urine,  sterilized  by  filteration  through  porcelain,  added  to  the 
mixture  of  blood-serum  and  agar,  improves  its  character,  ac- 
cording to  some  writers. 

Baer*  recommends  the  following  medium:  Hydrocele, 
pleuritic,  or  ascitic  fluid  should  be  caught  under  aseptic  con- 
ditions in  sterile  flasks,  distributed  into  test-tubes,  and  tested 
for  sterility  in  the  incubator  for  24  hours  at  37°  C.  This  is 
mixed  with  plain  agar  which  has  been  previously  condensed 
to  two-thirds  its  bulk  in  the  proportion  of  two  parts  agar  to  one 
of  the  transudate.  The  transudate  is  added  to  the  agar  in  tubes, 
the  agar  having  been  melted  and  cooled  to  45°  C.  The  tubes 
so  prepared  are  allowed  to  solidify  in  a  slanting  position,  cap- 
ped with  rubber  caps  which  have  been  sterilized  in  a  i-iooo 
corrosive  sublimate  solution,  and  then  placed  in  the  incubator 
again  for  two  days  to  test  their  sterility.  The  slant  tubes  may  be 
used  for  plating  out  the  gonorrheal  pus  by  taking  a  loopful  of 
the  material  to  be  examined  and  smearing  it  over  the  surface 
of  the  agar,  using  the  condensation  water  in  the  tube  to  assist 
in  the  spreading.  Other  organisms  identical  in  morphology 
and  in  staining  properties,  including  the  negative  Gram  reac- 
tion may  be  cultivated  from  suspected  gonorrheal  discharge, 
but  these  grow  on  ordinary  culture  media.  A  poison  has  been 
extracted  from  cultures  of  the  gonococcus  which  produces  toxic 
symptoms  in  various  animals. | 

The  colonies  of  the  gonococcus  are  very  small,  grayish- 
white,  circular,  translucent;  appearing  after  from  twenty-four 
to  forty-eight  hours.  They  may  attain  a  diameter  of  i  to  2  mm. 
The  gonococcus  will  occasionally  develop  on  ordinary  glycerin- 
agar  or  Loffler's  blood-serum  medium,  but  the  growth  is  likely 
to  be  feeble  and  cannot  be  relied  on.  The  cultures  live  for  a 

*Journ.  Infect.  Dis.     Vol.  IV.,  1904.  pp.  313-326. 

fNeisser  and  Scholtz  in  Kolle  and  Wassermann.     Bd.  III.,  1903.    p.  174. 


314  MANUAL   OF    BACTERIOLOGY. 

considerable  time  if  kept  from  drying.  The  gonococcus  is  not 
known  to  produce  urethritis  or  conjunctivitis  in  any  of  the  lower 
animals.  In  the  peritoneum  it  may  cause  suppurative  in- 
flammation in  mice  and  guinea-pigs.  Reproduction  of  the  dis- 
ease in  man  has  been  effected  by  experimental  inoculation 
with  pure  cultures.  Besides  being  the  cause  of  gonorrheal 
urethritis  and  infection  of  the  cervix  uteri,  the  gonococcus  has 
been  isolated  from  cases  of  vaginitis  in  little  girls  and  from 
gonorrheal  conjunctivitis.  It  has  been  found  to  be  the  cause 
of  many  cases  of  pyosalpinx,  as  well  as  of  gonorrheal  proc- 
titis,  naphritis,  arthritis,  myocarditis  and  endocarditis;  these 
conditions  complicating  gonorrhea  may  also  be  secondary  or 
mixed  infections. 

Bacillus  of  Soft  Chancre  (of  Ducrey).— A  small,  oval 
bacillus,  usually  occurring  in  chains.  It  stains  with  ordinary 
aniline  dyes,  but  not  by  Gram's  method.  It  has  been  culti- 
vated on  human  blood-agar  (also  rabbit  blood-agar;  the 
medium  deteriorates  in  a  few  weeks — Davis).  It  is  cultivated 
with  difficulty.  It  is  found  in  the  pus  of  soft  chancre  or  chan- 
croid, usually  mixed  with  other  organisms.  It  has  been  dem- 
onstrated in  sections  of  the  ulcers.  There  seems  to  be  un- 
certainty with  respect  to  its  occurrence  in  buboes.  Ducrey 
was  able  to  secure  it  in  pure  culture  by  successive  inoculations 
on  the  human  skin.  Although  this  bacillus  has  not  yet  been 
sufficiently  studied,  there  seems  little  doubt  that  it  is  the  cause 
of  soft  chancre.* 

Bacillus  Pneumoniae  (of  Friedlander),  or  Bacillus  mucosus 
capsulatus.^ — A  short  bacillus  with  rounded  ends,  sometimes 
growing  out  to  a  greater  length;  sometimes  occurring  in  pairs; 
surrounded  by  a  capsule  which  is  seen  only  in  preparations 
made  from  the  tissues  of  infected  animals,  and  is  not  seen  in 
cultures.  This  bacillus  is  not  motile.  It  does  not  form  spores. 

*Davis.     Journ.  Medical  Research.     Vol.  IX.     1903. 

fHoward.  Philadelphia  Medical  Journal.  February  19,  1908.  Curry 
Howard,  Perkins.  Journal  Experimental  Medicine.  Vols.  IV.  and  V. 


PATHOGENIC    BACTERIA.  315 

It  stains  with  the  ordinary  aniline  dyes,  but  does  not  stain  by 
Gram's  method.  It  is  aerobic  and  facultatively  anaerobic. 
It  may  be  cultivated  at  ordinary  temperatures,  but  grows 
best  at  high  temperatures.  It  does  not  liquefy  gelatin.  Stick- 
cultures  in  gelatin  develop  a  round,  flat  knob  at  the  point  where 
the  puncture  enters  the  surface  of  the  gelatin,  making  what  is 
called  a  anail-shaped"  growth;  the  growth  in  gelatin  is  white; 
in  old  cultures  the  various  media  acquire  a  brown  color. 
Dextrose  and  lactose  are  fermented  by  it;  in  cultures  on  potato, 
gas  is  formed,  causing  a  frothy  appearance;  milk  is  not  coagu- 
lated. It  does  not  produce  indol. 

The  thermal  death-point  is  about  56°  C.  moist  heat.  Strains 
of  this  organism  are  pathogenic  for  mice,  less  so  for  guinea-pigs 
and  rabbits.  This  bacillus  is  sometimes  found  in  the  healthy 
mouth  and  nose.  It  has  been  known  to  cause  inflammation, 
especially  in  the  eyes,  mouth,  nose  and  ear;  also  bronchopneu- 
monia,  and  more  rarely  empyema  and  meningitis.  It  was  de- 
scribed by  Friedlander  as  the  specific  cause  of  lobar  pneu- 
monia; but  more  recent  investigations  indicate  that  it  is  com- 
paratively seldom  found  in  this  disease. 

There  are  various  capsulated  bacilli  (capsule  bacilli  of  R. 
Pfeiffer  and  others)  which  closely  resemble  the  bacillus  of 
Friedlander,  and  at  least  belong  to  the  same  group.  The 
bacillus  of  ozena,  which  has  often  been  found  in  that  disease 
is  very  similar.  B.  lactis  aerogenes  and  B.  coli  communis  also 
have  many  points  in  common  with  the  Friedlander  bacillus. 

Perkins,*  as  a  result  of  his  studies  of  this  organism,  comes 
to  the  conclusion  that  there  is  no  one  organism  entitled  to  the 
name  exclusively,  but  that  the  term  includes  a  large  number  of 
organisms  which  have  been  given  various  names.  The  only 
method  of  differentiating  the  different  members  of  the  group  is 
by  noting  the  fermentation  reactions  with  sugars.  He  makes 
three  tentative  groups.  All  carbohydrates  fermented  with  the 

*Journ.  Infec.  Dis.     Vol.  I.     1904.     pp.  241-267. 


316  MANUAL    OF    BACTERIOLOGY. 

formation  of  gas;  all  carbohydrates  except  saccharose  fer- 
mented with  the  formation  of  gas:  all  carbohydrates  except  sac- 
charose fermented  with  the  formation  of  gas.  He  would  call 
the  first  group  Bacterium  aero  genes;  the  second,  Bacterium 
pneumonicum,  the  third,  Bacterium  acidi  lactici. 

Under  Bacterium  aerogenes  he  would  include  Bacterium 
aerogenes  already  so  called,  B.  capsulatus  septicus,  and  several 
cultures  with  various  names  obtained  from  different  sources : 
Several  from  Johns  Hopkins  University  labeled  "B.  pneum. 
Friedlander"  B.  hemorrhagic  septicemia  Howard,  B.  mucosus 
Blumer,  B.  mucosus  capsulatus  Wright  and  Mallory,  besides 
others. 

Under  Bacterium  pneumonicum  Friedlander,  B.  capsulatus 
Fashing,  B.  sputiginus  crassus,  B.  ozence,  and  probably  B. 
rhino  scleroma,  and  other  of  the  Johns  Hopkins  cultures. 

Under  Bacterium  acidi  lactici  he  would  include  the  organism 
going  by  this  name. 

Infections  due  to  this  organism  are  very  prevalent  in  Perkins 
locality  (Cleveland,  O.),  and  are  caused  almost  exclusively  by 
members  of  the  Boot,  aerogenes  division. 

Bacillus  of  Rhinoscleroma.* — A  short  bacillus  with  rounded  ends,  often 
united  in  pairs,  also  growing  to  a  greater  length;  surrounded  by  a  capsule;  not 
motile;  stained  by  the  ordinary  aniline  dyes.  It  is  much  like  the  bacillus  of 
Fiiedlander,  but  some  writers  state  that  it  retains  Gram's  stain  more  tena- 
ciously than  that  organism;  this  may  be  doubted,  however.  The  organism 
has  been  cultivated.  It  is  a  facultative  anaerobe.  It  grows  rapidly,  best  in 
the  incubator.  It  does  not  liquefy  gelatine;  its  growth  in  gelatin  stick-cultures, 
resembles  that  of  the  bacillus  of  Friedlander.  It  grows  on  the  ordinal y  media. 
Gas  may  be  developed  upon  potato. 

It  is  pathogenic  for  mice  and  guinea-pigs,  less  so  for  rabbits.  Its  virulence 
is  less  than  that  of  Friedlander's  bacillus. 

It  has  been  obtained  from  the  tissues  of  cases  of  rhinoscleroma.  Rhino- 
scleroma  is  a  disease  characterized  by  a  chronic  tubercular  thickening  and 
swelling  of  the  skin  around  the  nose  and  similar  swelling  of  the  nasal  mucous 


*Perkins  comes  to  the  conclusion  from  his  investigations  that  this  organism 
has  no  etiological  connection  with  the  disease  in  question,  but  that  it  is  rather 
a  secondary  invader.  Journ.  Inf.  Dis.  Vol.  IV.,  No.  i.  p.  65. 


PATHOGENIC    BACTERIA.  317 

membrane,  sometimes  followed  by  ulceration.     It  is  commonest  in  Austria  and 
Italy.     It  has  been  seen  in  America  only  very  rarely. 

The  organisms  may  be  stained  in  the  diseased  tissues,  but  their  detection  is 
a  matter  of  considerable  difficulty,  and  they  are  not  always  found.  It  is  not 
yet  certain  that  they  are  the  cause  of  rhinoscleroma. 

Bacillus  Mycogenes.* — A.  plump,  short  bacillus,  less  than 
i  /x  in  breadth,  possessing  no  flagella,  non-motile,  does  not 
form  spoes;  capsules,  are  seen  in  preparations  from  tissues  of 
inoculated  animals  and  in  milk  cultures,  rarely  in  preparations 
from  agar  cultures.  The  organism  occurs  singly  or  in  pairs, 
and  even  in  longer  filaments.  Gram  positive  in  tissues,  but 
negative  in  cultures. 

The  growth  on  agar  is  porcelain  white  and  viscid.  In  all 
liquid  media  viscidity  is  very  marked.  Gelatin  is  not  liquefied. 
Coagulated  blood-serum  not  liquefied.  "Nail-head"  growth 
shows  in  stab  cultures.  Milk  is  coagulated  in  one  to  five  days. 
Casein  not  digested.  Litmus  is  reduced.  Growth  on  potato 
is  brown  and  slimy,  but  there  is  no  gas  formation.  Indol 
negative.  None  of  the  sugars  are  fermented. 

Very  pathogenic  for  rabbits  and  guinea-pigs.  Rabbits  are 
killed  in  eighteen  hours  by  subcutaneous  injection  of  T^Q-  c.c. 
of  a  twenty-four  hour  beef-broth  culture,  guinea-pigs  in  less 
than  fifteen  hours  by  the  same  dose. 

Bacillus  Pyocyaneus. — A  slim  bacillus  with  rounded  ends. 
It  is  motile.  It  does  not  form  spores.  It  is  decolorized  by 
Gram's  method.  It  is  aerobic;  grows  well  at  ordinary  tem- 
peiatures;  liquefies  gelatin,  and  grows  on  the  ordinary  culture- 
media.  Cultures  persent  a  blue  or  green  color,  especially  in 
transparent  media.  This  color  is  not  confined  to  the  growth 
itself,  but  a  blue  or  green  fluorescence  spreads  over  the  whole 
medium.  In  an  old  agar  culture  the  color  may  become  very 
dark.  The  pigment  forms  in  the  presence  of  oxygen,  and  is 
due,  at  least  in  part,  to  the  ptomaine,  pyocyanin.  On  potato 

*Ralph  T.  Edwards.   Journal  oj  Injections  Diseases.   Vol.  II.,  No.  3.    1905. 


318  MANUAL   OF    BACTERIOLOGY. 

the  growth  is  usually  brown;  the  surrounding  medium  may 
be  tinged  with  green.  Milk  is  coagulated  and  peptonized 
and  an  acid  reaction  is  developed.  Indol  is  formed  in  Dun- 
ham's peptone  solution.  Coagulated  blood-serum  is  liquefied. 
The  Bacillus  pyocyaneus  seems  to  be  rather  widely  dis- 
tributed in  nature;  it  has  been  found  on  the  skin,  in  normal 
feces,  also  in  diarrheal  discharges  and  in  dysentery.  It  is  the 


*          Vs  *  *  -  K> 

'    1    •      <          "a    U      >*; 


FIG.  75. — Bacillus  pyocyaneus  pure  culture.     (X  1000.) 

cause  of  the  color  in  blue  or  green  pus.  It  has  frequently  been 
demonstrated  in  pus,  but  oftenest  perhaps,  in  mixed  infections. 
It  has  been  found  in  various  abscesses,  in  otitis  media,  peritoni- 
tis, appendicitis  and  bronchopneumonia.  It  has  been  known 
to  produce  general  septicemia.*  It  is  pathogenic  for  guinea- 
pigs  and  rabbits,  in  whom  it  may  produce  septicemia.  In 
animals  it  may  lead  only  to  local  suppuration,  from  which  they 

*Lartigau.  Philadelphia  Medical  Journal.  September  17,  1898.  Journal 
Experimental  Medicine.  Vol.  III.  1898.  Perkins.  Journal  of  Medical  Re- 
search. Vol.  VI.  1901. 


PATHOGENIC    BACTERIA.  319 

may  recover,  being  made  immune  from  subsequent  infection 
with  this  organism. 

Emmerich*  has  obtained  a  bacteriolytic  enzyme  by  passing 
cultures  of  B.  pyocyaneus  three  weeks  old  through  Berkefeld 
niters.  To  this  substance  he  has  given  the  name  "pyo- 
cyanase,"  and  he  finds  that  it  not  only  disintegrates  the  bacillus 
pyocyaneus  itself,  but  also  the  bacteria  of  cholera,  diphtheria, 
typhoid,  plague,  anthrax,  also  streptococci,  staphylococci,  and 
gonococci.  Tubercle  bacilli  and  the  hay  bacilli  are  not  af- 
fected by  the  enzyme.  The  substance  has  been  used  with 
very  favorable  results  not  only  in  experiment  upon  animals, 
but  also  in  human  diphtheria^  not  only  in  dissolving  the  mem- 
brane in  the  throat  in  these  cases  but  in  neuralizing  the  toxin  of 
the  disease.  Reports  on  all  sides  seem  so  far  very  favorable  to 
its  use  as  a  therapeutic  agent. 

There  appears  to  be  a  whole  group  of  fluorescent  organisms 
of  slightly  different  characters  which  closely  resemble  one 
another,  all  classed  as  pyocyaneus.  - 

Bacillus  Proteus. — A  bacillus  with  rounded  ends,  varying 
much  in  length,  breadth  0.4  to  0.6  /*;  frequently  appearing  as 
short  ovals  like  micrococci;  sometimes  growing  out  into  long 
filaments,  so  that  it  is  said  to  be  pleomorphic.  Rounded  in- 
volution forms  occur.  It  is  not  stained  by  Gram's  method. 
It  is  motile.  Spore  formation  has  not  been  observed.  It  is 
aerobic  and  facultatively  anaerobic.  It  grows  rapidly  at  or- 
dinary temperatures.  This  organism  was  originally  described 
by  Hauser  as  three  different  species — Proteus  vulgaris,  which 
was  said  to  liquefy  gelatin  rapidly,  Proteus  mirabilis,  which 
liquefied  gelatin  slowly,  and  Proteus  Zenkeri,  which  did 
not  liquefy  gelatin.  It  seems  probable  that  these  organisms 
were,  in  fact,  varieties  of  the  same  species,  now  called  Bacillus 
proteus.  To  proteus  vulgaris  or  some  closely  allied  form  has 

*Munchner  Med.  Wochenschr.  Nov.  5,  1907.  Vol.  LIV.,No.  45.  Cited  in 
Journ.  Am.  Med.  Assn.  Dec.  14,  1907. 


320 


MANUAL    OF    BACTERIOLOGY. 


been  attributed  the  causation  of  several  cases  of  poisoning  from 
spoiled  meat.*  Upon  gelatin-plates  the  colonies  present  a 
characteristic  phenomenon,  when  seen  under  the  low  power,  in 
the  projection  of  processes  which  subsequently  change  their 
form  and  position,  and  which  may  become  entirely  detached 
from  the  original  colony,  so  that  the  surface  of  the  gelatin  may 
become  covered  with  so-called  "swarming  islands." 

The  proteus  grows  on  the  usual  media,  tending  to  produce  a 
foul  odor,  decomposition  and  alkaline  reaction.  In  urine  it 
converts  urea  into  ammonium  carbonate. 

This  organism  is  one  of  those  which  were  formerly  described 
under  the  name  of  Bacterium  termo.  It  is  among  the  most 
common  and  widely  distributed  bacteria.  It  has  been  found 
in  decomposing  animal  and  vegetable  substances,  in  the 
feces,  in  the  urine  in  cystitis  and  in  the  discharges  of  children 
suffering  from  cholera  infantum.  It  appears  that  this  organ- 
ism may  occasionally  be  pathogenic  to  man,  causing  pus  forma- 
tion, peritonitis  and  even  general  infection.!  Cultures  in- 
jected in  considerable  amounts  may  be  pathogenic  to  animals. 

Bacillus  of  Bubonic  Plague.  (Bacillus  sive  Bacterium 
Pestis  Bubonicae). — An  oval  or  short  rod-shaped  bacillus,  with 
rounded  ends,  sometimes  possessing  a  capsule.  It  occurs 
singly  or  in  pairs  rarely  in  chains.  Involution  forms  are 
met  with  in  material  from  old  buboes.  Branching  forms  have 
been  noted.  J  It  is  not  motile.  It  does  not  form  spores. 
With  the  aniline  dyes  the  ends  stain  more  deeply  than  the 
middle;  this  is  called  polar  staining;  by  Gram's  method  it 
is  decolorized.  It  is  aerobic.  It  grows  at  ordinary  tem- 
peratures, but  better  in  the  incubator.  It  grows  on  most 
media.  The  growths  are  grayish-white.  Gelatin  and  blood- 
serum  are  not  liquefied.  In  bouillon,  the  medium  remains 
clear,  while  a  granular  deposit  forms  on  the  sides  and 

*Giinther  Loc.  cit.     p.  709.  . 

tWare.     Annals  of  Surgery.     Vol.  XXXVI.     1902. 

|Kolle.     Zeitschr.  f.  Hygiene,     Bd.  36.     1901.     p.  399. 


PATHOGENIC    BACTERIA.  32! 

bottom  of  the  tube.  In  bouillon  to  which  a  few  minute  drops 
of  sterile  oil,  as  cocoanut  oil,  have  been  added,  a  growth  takes 
place  from  the  under  side  of  the  oil  drops.  Such  growths  ex- 
tend down,  and  are  called  stalactite  growths.  The  stalactites 
break  off,  with  the  slightest  disturbance. 

Remarkable  involution  forms  appear  on  agar  containing 
3  per  cent,  of  common  salt.  The  stalactite  growths  and  the 
forms  occurring  on  salt-agar  are  considered  the  most  charac- 
teristic cultural  peculiarities.* 


*-r 

FIG.  76.— Bacillus  of  bubonic  plague. — (Yersin.) 

It  is  sometimes  sensitive  to  drying,  but  may  sometimes  sur- 
vive prolonged  drying.  When  spread  in  thin  layers,  it  is  killed 
in  three  to  four  hours  by  direct  sunlight.  Dried  out  on  cotton 
and  linen  cloth  the  bacilli  were  found  to  be  still  alive  after  18 
hours  exposure  to  sunlight.  The  action  of  sunlight  is  in  direct 
proportion  to  the  thinness  of  the  layer  into  which  the  cultures 
are  spread,  being  seriously  hindered  when  the  layer  is  thick, 
in  a  few  minutes  by  steam  at  100°  C.  Rosenau  states  that  it 
is  killed  in  one  hour  by  one  per  cent,  carbolic  acid,  but  others 

.     *  Wilson.     Journal  Medical  Research.     Vol.  VI.     1901. 
21 


322 


MANUAL   OF    BACTERIOLOGY. 


state  that  it  requires  longer  than  this.  Kolle  and  Wasser- 
mann  quote  various  authorities,  none  seem  to  agree.  Abel  states 
that  it  required  two  hours  in  this  strength.  Gioxa  and  Gosio, 
three  hours.  In  solutions  of  higher  strength  the  discrepancy 
between  authorities  varies  also;  in  5  per  cent,  solution  from 
one  to  ten  minutes,  one  hour  in  i  per  cent,  carbolic  acid.* 
It  is  pathogenic  for  rats,  mice,  guinea-pigs,  rabbits  and  a  num- 
ber of  other  animals  besides  flies  and  other  insects.  The 
rat-flea,  pulex  cheapis,  has  been  shown  by  the  British  Commis- 
sion! to  bite  human  beings;  the  inference  from  this  is  that  the 
plague  bacillus  is  conveyed  from  the  rat  to  man  in  this  way. 
Thompson!  holds  that  the  ftea  is  the  intermediary  between 
the  plague  rat  and  human  beings; 

In  man  it  appears  usually  to  enter  through  wounds  of  the 
skin.  Other  possible  avenues  of  infection  are  the  air-passages, 
the  mouth  and  the  gastro-intestinal  tract.  In  plague  three 
different  clinical  forms  are  to  be  recognized — the  bubonic, 
the  pneumonic  and  the  septicemic.  The  bubonic  form  is  com- 
monest. The  point  in  the  skin  at  which  the  inoculation  takes 
place  seems  generally  to  exhibit  no  inflammatory  reaction. 
The  lymph-nodes  are  generally  swollen,  especially  the  deep  in- 
guinal and  axillary  nodes.  The  swollen  lymph-nodes  may 
suppurate.  The  suppurating  nodes  are  often  infected  simul- 
taneously with  micrococci.  The  bacilli  are  numerous  in  the 
enlarged  lymph-nodes,  but  may  be  detected  in  the  other  organs 
of  the  body  and  in  the  blood.  The  organism  is  furthermore 
to  be  found  in  the  fluid  aspirated  from  buboes  during  life. 
It  may  be  cultivated  from  this  fluid,  and  recovered  from  rats 
and  guinea-pigs  inoculated  with  it.  In  the  pneumonic  or  pul- 


*Rosenau.      Viability  of  Bacillus  pestis.     Marine  Hospital  Service.       Hy- 
gienic Laboratory  Bulletin.     No.  4.     190 1. 

fLondon  letter  to  Journ.  Am.  Med.  Assn.     Nov.  16,  1907.      See  also  Ibid. 
Vol.  L.,  No.  2.     Jan.  n,  1908.     p.  127. 

"[Australian  Medical  Gazette.     Nov.  20,  1907.    Abs.  Journ.  Am.  Med.  Assn. 
Jan.  18,  1908.     p.  127. 


PATHOGENIC    BACTERIA.  323 

monary  form  the  bacilli  occur  in  the  sputum,  and  may  be 
tested  in  the  same  manner.  This  type  of  the  disease  is  said  to 
be  very  fatal.  In  the  septicemic  form  no  primary  bubo  is 
found;  but  a  bubonic  case  may  become  septicemic,  and  this 
form  is  also  very  fatal. 

During  epidemics  of  plague  it  has  been  noted  that  rats  may 
die  in  large  numbers,  and  plague  bacilli  have  often  been  re- 
covered from  the  bodies  of  such  rats.  The  systematic  de- 
struction by  health  departments  of  all  the  rats  possible  is  im- 
portant where  an  epidemic  is  present  or  is  feared.  The  same 
applies  to  mice.  The  agency  of  fleas  as  carriers  of  the  bacilli 
has  been  suggested,  and  according  to  Thompson*  this  has  been 
proven.  Flies  have  also  been  suggested  as  carriers. 

No  one  but  the  most  experienced  and  strictly  careful  person 
should  trust  himself  with  experiments  with  cultures  of  the 
plague  bacillus.  The  danger  is  so  great,  not  only  to  the  worker 
himself,  but  to  those  around  him  that  the  cultures  should  be 
handled  in  a  room  where  only  the  worker  himself  is  engaged  in 
his  experiments.  It  follows',  of  course,  that  cultures  should 
under  no  circumstances  be  entrusted  to  classes  of  students  un- 
der instruction. 

The  greatest  care  must  be  used  in  working  with  the  bacillus 
of  plague.  A  number  of  fatal  results  have  occurred  through 
it  in  laboratory  investigators. 

Haffkine  has  devised  a  method  of  protective  inoculation 
against  plague  consisting  of  the  injection  of  small  doses  from 
cultures  in  which  the  bacilli  have  been  killed.  An  accidental 
infection  with  tetanus  at  the  time  of  injection  in  19  persons 
in  India  rather  discredited  this  method  for  a  while,  but  sub- 
sequent results  have  been  very  encouraging.  An  active  im- 
munity which  is  quite  lasting,  it  is  maintained,  may  be  secured 
by  this  method  in  some  days.  The  injection  is  sometimes 

*J.  A.  Thompson.  Australian  Medical  Gazette.  Sidney,  Australia.  Nov. 
20,  1908. 


324 


MANUAL   OF    BACTERIOLOGY. 


followed   by    considerable   constitutional    disturbance.      This 
method  seems  likely  to  be  of  considerable  value. 

Yersin  and  others  have  prepared  protective  sera  on  the  same 
general  principles  used  in  making  other  sera  for  effecting  pas- 
sive immunity.  The  results  so  far  obtained  with  these  sera 
are  not  especially  encouraging.* 


FIG.  77. — Bacillus  aerogenes  capsulatus,  smear  preparation  from 
rabbit's  liver..     (X  1000.) 


An  agglutination  reaction  has  been  described;  but  this  is  not 
likely  to  be  of  great  value  in  diagnosis. 

The  period  of  incubation  in  this  disease  is  from  two  to  seven 
days.  It  has  occasionally  appeared  in  civilized  countries  dur- 
ing recent  times,  though  not  to  a  very  serious  extent.  Among 
the  localities  of  importance  to  us  it  has  recently  visited  the 
Philippine  Islands,  California  and  Mexico.  It  has  ravaged 

*Gimther.     Loc.  cit.     p.  630. 


PATHOGENIC    BACTERIA.  325 

the  southeastern  part  of  Asia  within  a  few  years.  In  the 
Middle  Ages,  and  in  succeeding  centuries,  it  devastated  many 
of  the  countries  of  Europe,  where  it  was  one  of  the  most  im- 
portant of  the  pestilences  that  went  in  those  days  by  the  name 
of  the  "plague."  It  appears  to  have  been  the  disease  known 
in  English  history  as  the  "black  death."* 

Paraplague  Bacillus. — Neumann!  described  an  organism 
which  he  isolated  from  rats  in  Hamburg  which  differs  from  B. 
pestis  bubonicas  only  in  that  it  produces  no  results  when  in- 
jected subcutaneously  or  intraperitoneally  into  rats  and,  further- 
more, in  that  agglutination  'tests  were  negative.  Breathing 
into  the  lungs  of  very  minute  quantities  was  fatal  for  rats. 

Bacillus  Aerogenes  Capsulatus. — A  thick  bacillus,  3  to  6 
/*  in  length,  frequently  capsulated,  discovered  by  Welch  and 
Nuttall.  The  capsules  may  be  found  in  preparations  from 
animal  tissues,  but  rarely  in  cultures.  It  sometimes  forms 
spores,  chiefly  in  cultures  on  blood-serum.  The  vegetative 
forms  are  destroyed  at  58°  C.  moist  heat  in  ten  minutes,  but  the 
spores  withstand  boiling  nearly  eight  minutes.  It  is  not  mo- 
tile. It  stains  by  Gram's  method.  It.  is  anaerobic,  and  is 
readily  cultivated  by  Buchner's  method  for  anaerobes.  It 
grows  best  at  the  body  temperature,  but  will  grow  at  the  room 
temperature.  It  may  liquefy  gelatin  slowly  or  not  at  all.  The 
growths  are  whitish.  In  media  containing  lactose,  dextrose  or 
saccharose  it  produces  an  abundance  of  gas;  but  according  to 
Welch,  it  is  also  able  to  form  gas  from  proteids.  Milk  is  co- 
agulated, and  the  reaction  becomes  acid.  Gas  forms  upon 
potato,  where  the  growth  is  thin  and  grayish-white. 

It  occurs  in  the  intestine  of  man  and  various  other  animals, 
in  soil,  sewage  and  water.  It  is  not  usually  pathogenic  for 
rabbits  and  mice.  In  guinea-pigs,  sparrows  and  pigeons  it  may 

*For  further  details  concerning  plague  consult  articles  by  Parker,  Novy 
and  Flexner.  Transactions  o}.  the  Association  American  Physicians.  1902. 
Calvert.  American  Medicine.  January  24,  1903. 

~\Zeitschr.  }.  Hygiene.     XLV.,  No.  5.     1903. 


326 


MANUAL    OF    BACTERIOLOGY. 


produce  "gas  phlegmons."  It 
has  been  found  on  numerous 
occasions  in  the  organs  of  human 
cadavers  in  which  a  development 
of  gas  had  taken  place,  produc- 
ing bubbles  or  cavities  in  the 
tissues,  imparting  to  them  a 
peculiar  spongy  character  (Ger- 
man, Schaumorgane).  Probably 
this  is,  as  a  rule,  a  postmortem 
invasion,  but  there  is  reason  to 
believe  that  in  some  cases  it 
enters  the  circulation  during  life. 
It  has  been  found  in  cases  of 
emphysematous  gangrene  or  cel- 
lulitis,  in  various  uterine  infec- 
tions, including  physometra  and 
emphysema  of  the  uterine  wall, 
in  pneumothorax  and  pneumo- 
peritonitis,  and  in  other  patho- 
logical conditions  where  gas 
occurs  in  the  tissues.  Excep- 
tionally it  may  cause  pus-forma- 
tion.* This  bacillus,  or  the  gas 
formed  by  it  in  the  organs  of 
human  cadavers,  appears  to  have 
furnished  the  basis  for  some  of 
the  cases  in  which  death  has  been 
ascribed  to  the  entrance  of  air 
into  the  veins  during  life.  It  is 
FIG.  78.— Bacillus  aerogenes  cap-  the  same  as  the  organism  de- 
gal-bubWes.  dextro8w^'  scribed  by  E.  Frankel  as  Bacillus 
phlegmonis  emphysematosae. 

*  Welch.     Philadelphia  Medical  Journal.     August  4,  1900. 


PATHOGENIC    BACTERIA.  327 

Bacillus  Edematis  Maligni  (French,  Vibrion  septique).—A 
bacillus  about  i  M  in  breadth,  2  to  10  M  in  length,  which  may 
form  threads,  having  rounded  ends  when  occurring  singly.  It 
is  motile,  having  flagella  at  the  sides  and  ends.  It  forms 
spores,  and  may  bulge  at  the  center  in  consequence  of  the 
spores  formed  there.  It  retains  the  stain  by  Gram's  method. 
It  is  a  strict  anaerobe.  It  grows  at  ordinary  temperatures,  but 
better  in  the  incubator.  It  liquefies  gelatin  and  blood-serum. 
The  colonies  in  gelatin  are  spherical  and  appear  like  little 
bubbles.  It  grows  well  upon  agar.  Gas  may  be  produced  in 
these  media. 

It  is  found  in  garden-earth,  street  dirt  and  in  putrefying 
organic  material.  It  is  pathogenic  to  rabbits,  guinea-pigs, 
mice,  pigeons  and  various  other  animals,  including  man.* 
Inoculation  results  in  the  production  of  swelling  and  edema, 
spreading  from  the  point  of  inoculation.  Gas  may  be  pro- 
duced in  the  tissue.  It  may  lead  to  wide-spread  septicemia. 

Bacillus  Tetani. — A  slim,  straight  bacillus,  with  rounded 
ends,  which  may  form  in  threads.  It  is  slightly  motile. 
Spores  form  in  culture- media  at  the  end  of  thirty  hours  in  the 
incubator.  The  spores  are  usually  round  though  it  is  stated 
that  they  are  egg-shaped  when  grown  on  media  containing 
much  sugar  or  on  rice,  located  at  one  end,  which  is  swollen,  so 
that  in  this  stage  the  organism  has  the  shape  of  a  drum-stick. 
The  spores  are  extremely  resistant,  and  in  the  dry  condition 
remain  capable  of  germinating  under  favorable  conditions  for 
years. 

Earlier  statements  in  regard  to  the  destruction  of  tetanus 
spores  by  steam  have  generally  placed  the  length  of  exposure 
much  too  short.  Among  others,  Theobald  Smithf  found  the 
spores  much  more  resistant  to  sterilization  by  steam  than  the 
statements  usually  made.  He  found  that  in  some  cases  the 

*  Gould.     Am.  Surgery.     October,  1903. 

•\Journ.  Am.  Med.  Assn.     March  21,  1908.     pp.  929-934. 


328  MANUAL   OF    BACTERIOLOGY. 

tetanus  spores  in  his  cultures  resisted  boiling  40  to  70  minutes. 
This  is  of  importance  from  the  fact  that  the  tetanus  spores  are 
so  generally  distributed  in  our  environment,  and  that- in  dres- 
sings for  wound,  in  gelatin  used  for  wounds  particularly  the 
danger  of  not  properly  sterilizing  these  is  imminent.  The  sub- 
cutaneous injection  of  gelatin  to  check  obstinate  hemorrhage 
has  been  not  infrequently  followed  by  tetanus.  Tetanus  spores 
were  found  by  Falcioni*  to  resist  two  and  one-half  hours,  but 


«. 


FIG.  79. — One  tetanus  bacillus  (X  loco)  with  pus  cocci.     Preparation 
prepared  from  a  fatal  case  of  tetanus  in  a  human  being. 

not  three  hours  steaming  in  a  Koch  sterilizer.  The  spores  were 
exposed  in  this  case  in  2,  5,  and  10  per  cent,  gelatin  solutions. 
The  tetanus  bacillus  stains  by  Gram's  method.  It  is  a  strict 
anaerobe;  it  grows  in  an  atmosphere  of  hydrogen,  but  not  of 
carbon  dioxide.  It  may  sometimes  be  made  to  grow  very  well 
by  Buchner's  method.  It  may  be  cultivated  at  the  room  tem- 
perature, but  better  in  the  incubator.  It  grows  upon  ordinary 

*Annali  d'Igiene  Sperimental.     1904.     Quoted  by  Smith,  loc.  cit. 


PATHOGENIC    BACTERIA.  329 

culture- media,  perferably  those  containing  dextrose.  Gelatin 
is  liquefied  slowly;  the  colonies  in  gelatin  present  characteristic 
radiating  filaments  and  look  like  a  bristle  brush.  It-  grows  on 
the  other  culture-media.  Gas  formation  is  not  pronounced. 

This  organism  appears  to  be  widely  spread  in  external 
nature,  especially  in  the  soil.  It  is  often  found  in  garden- 
earth  and  in  the  feces  of  herbivorous  animals.  It  is  conse- 
quently more  apt  to  be  encountered  in  practice  in  deep,  pene- 
trating wounds  caused  by*dirty  nails  and  the  like.  McFar- 
land  claims  that  it  may  occur  in  vaccine  virus  when  this  is 
carelessly  prepared,  and  this  would  explain  those  rare  cases  of 
tetanus  which  occur  after  vaccination.*  Tetanus  bacilli  have 
been  found  in  gelatin,  and  it  is  stated  that  tetanus  has  followed 
the  injection  of  gelatin  as  a  hemostatic.  The  infection  ap- 
pears usually,  if  not  always,  to  be  introduced  through  some 
wound. t  Clinically,  persons  having  the  disease  suffer  from 
spasms  of  the  muscles  about  the  neck  and  the  lower  jaw  (lock- 
jaw). The  spasms  finally  become  general. 

Inoculation  with  a  pure  culture  produces  tetanus  in  a  great 
many  animals.  Horses  and  guinea-pigs,  are  very  susceptible, 
mice,  rabbits,  rats  are  less  so  in  the  order  named.  Sheep,  dogs, 
pigeons  and  chickens  are  but  little  susceptible.  The  tetanic 
spasms  begin  in  the  vicinity  of  the  point  of  inoculation  and 
afterward  become  general.  The  bacilli  are  not  widely  scat- 
tered through  the  body;  they  occur  only  in  the  immediate 
vicinity  of  the  original  lesion,  and  there  are  no  important  mac- 
roscopic alterations  in  the  internal  viscera. 

Tetanus  is  the  type  of  the  purely  toxic  disease.  Its  symp- 
toms may  be  produced  in  animals  by  the  injection  of  liquid 
cultures  which  have  been  deprived  of  their  bacteria  by  fil- 
tration. The  toxic  substance  appears  not  to  be  a  ptomaine, 
as  was  at  first  supposed,  and  its  exact  nature  is  not  determined. 

*  Journal  Medical  Research.     Vol.  VII.     1902. 

t  Wells      Fourth  of  July  Tetanus.     American  Medicine.     June  13,  1903 


330  MANUAL    OF    BACTERIOLOGY. 

Cernovodeanu  and  Henri*  found  that  the  toxin  is  conveyed 
by  the  blood.  For  if  the  veins  of  the  part  are  cut,  the 
occurrence  of  symptoms  is  retarded  to  the  same  extent  as  by 
section  of  the  nerve.  After  section  of  the  vein  and  ligation  of 
the  muscles,  tetanus  toxin  can  be  injected* into  the  limb  with- 
out causing  tetanus.  That  part  of  the  sciatic  nerve  lying  in 
the  extremity  of  the  limb  in  which  the  veins  and  muscles 
are  tied  absorbs  the  toxin. 

The  poison  is  tremendously  powerful  (see  page  189).  It 
acts  as  an  excitant  to  the  motor  cells  of  the  central  nervous 
system,  especially  the  spinal  cord.  Bolton  and  Fisch  have 
shown  that  horses  used  for  the  preparation  of  diphtheria  anti- 
toxin may  be  infected  with  tetanus,  and  have  tetanus  toxin  in 
the  bloodf  even  before  symptoms  of  tetanus  are  observed  in 
the  horse. 

The  activity  of  the  poison  is  destroyed  by  heat  and  by  direct 
sunlight;  various  chemicals  diminish  its  intensity. 

As  shown  by  Noguchi,J  the  effects  of  certain  photodynamic 
substances  upon  ferments,  toxins,  and  protoplasma  have  been 
quite  extensively  studied.  Flexner  and  Noguchi§  repeated 
and  extended  the  work  of  others  in  this  direction  upon  tetanus 
toxin,  and  found  that  eosin  in  the  strength  of  i  per  cent,  solu- 
tion destroys  tetanospasmin  quickly  in  the  dark.  When  eosin 
and  tetanus  toxin  were  injected  at  different  places  in  the  body 
but  simultaneously  delayed  the  onset  of  the  symptoms  and 
porlonged  the  course  of  the  disease  though  it  did  not  actually 
prevent  the  fatal  termination.  Noguchi||  found  that  the  in- 
jection of  eosin  "gelb"  is  more  powerful  than  eosin  "rein." 
The  first  effect  on  the  organism  is  to  suppress  sporulation  and 

*Comptes  Rend.     Soc.  Biol.     V.  XLIL     p.   812.     May  4,    1907.     Abst. 
Bull  de  I'Inst.  Past.     Vol.  V.,  No.  18,  Sept.  30,  1907.     p.  804. 
f  Transactions  oj  the  Association  American  Physicians.      1902. 
iJourn.  Eocper.  Med.     Vol.  I.     1906.     pp.  252-267. 
<§Journ.  Exper.  Med.     Vol.  VIII.     1906.     pp.  1-7. 

'\\Loc.  ciL     Vol.  No.   3.     May  25,    1907.     pp.  291-297.    -Eben.     pp.  281- 
290. 


OF 


>       OF    THE  \ 

UNIVERSITY^ 

"    PATHOGENIC   BACTERIA.  331 

to  increase  thread  formation.  The  toxin-producing  power  and 
virulence  of  the  tetanus  bacillus  is  not  permanently  effected  by 
contact  for  a  long  period  with  eosin,  and  cultivation  in  media 
containing  eosin  does  not  effect  these  properties  permanently. 
Eosin  prevents  the  germination  of  spores  in  the  animal  body, 
and  it  causes  the  bacilli  to  degenerate  and  disappear  when  in- 
jected repeatedly  at  the  seat  of  the  tetanus  inoculation.  Un- 
germinated  tetanus  spores  remain  alive  at  the  point  of  in- 
jection in  eosin-treated  animals,  and  may  germinate  when  in- 


FI-.  80. — Anthrax  bacilli,  from  a  pure  culture.*     (X  1000.) 

troduced  into  a  different  location  in  the  same   animal   and 
cause  tetanus. 

Antitoxin  for  tetanus  has  been  prepared  according  to  the 
principles  employed  for  antitoxins  in  general.  It  has  not 
proved  very  markedly  successful  as  a  curative  agent;  but  as  a 
prophylaxis,  where  all  patients  are  treated  who  have  deep,  dirty 
wounds,  and  in  a  similar  way  in  veterinary  practice,  it  has  un- 
doubtedly proved  of  value.  Unfortunately  the  disease  is 
seldom  suspected  until  a  relatively  large  amount  of  toxin  has 

*  The  culture  was  derived  from  a  case  of  malignant  pustule  in  a  tanner. 
The  lesion  was  excised  promptly,  and  the  patient  recovered. 


332  MANUAL    OF    BACTERIOLOGY. 

formed  and  begun   to   manifest   its   action   in   the   patient's 
body.* 

Bacillus  Anthracis.— This  is  the  largest  of  the  pathogenic 
bacteria  with  the  exception  of  the  spirillum  of  relapsing  fever, 
which  is  longer  but  more  slender.  The  bacillus  of  anthrax 
is  about  1.25  /*  broad,  and  from  3  to  10  /*  long.  Bacillus 
aerogenes  capsulatus  is  of  about  the  same  size.  The  anthrax 
bacillus  often  forms  long  chreads.  A  capsule  is  sometimes 


FIG.  81. — Anthrax  bacilli,  showing  spores.     (X  1000.) 

present.  It  is  not  motile.  It  forms  spores,  which  are  placed 
in  the  centers  of  the  bacilli.  The  spores  form  only  in  the  pres- 
ence of  oxygen;  they  do  not  appear  in  the  body  of  an  infected 
animal  during  life.  Anthrax  spores  are  the  most  resistant  of 
all  pathogenic  bacteria;  they  have  been  known  to  withstand 
boiling  for  more  than  half  an  hour  f,  5  per  cent,  carbolic  acid 

*  Moschkowitz.  Studies,  Department  Pathology.  College  Physicians  and 
Surgeons.  New  York.  Vol.  VII.  1899-1900.  Annals  of  Surgery,  p.  442. 
1900. 

fV.  A.  Moore.     Infectious  Diseases  0}  Animals      1906. 


PATHOGENIC    BACTERIA.  333 

for  forty  days,  and  i-iooo  bichloride  of  mercury  for  nearly 
three  days.  The  anthrax  bacillus  is  aerobic,  although  not 
strictly  so.  It  stains  by  Gram's  method.  It  grows  at  the 
room  temperature,  but  better  in  the  incubator.  It  liquefies 
gelatin  and  coagulated  blood-serum.  Colonies  in  gelatin  seen 
under  a  low  power  display  numerous,  irregular,  fine,  hair-like 
projections;  stab-cultures  in  gelatin  also  present  fine  projec- 
tions passing  from  the  needle-puncture  into  the  solid  gelatin. 
It  grows  on  the  ordinary  culture-media;  the  growths  are  usually 


FIG.  82. — Colony  of  anthrax  bacilli  (low  power),  from  an  impression 
preparation  stained  with  methplene  blue. 

whitish.  Cultures  on  potato  kept  in  the  incubator  are  favor- 
able to  the  development  of  spores.  Milk  is  coagulated  and 
and  later  peptonized. 

It  is  pathogenic  for  mice,  guinea-pigs,  rabbits,  cats,  certain 
carnivora  and  a  great  many  other  animals;  it  is  also  specially 
pathogenic  for  sheep  and  cattle.  Rats  and  pigeons  are  quite 
resistant,  but  not  entirely  immune;  dogs  and  frogs  are  not  sus- 
ceptible, or  but  slightly  so. 

Anthrax  is  a  disease  which  occurs  spontaneously  chiefly  in 

cattle  and  sheep.     It  is  commoner  on  the  continent  of  Europe 

'and  in  Siberia  than  in  America.     In  susceptible  animals  in- 


334  MANUAL    OF    BACTERIOLOGY. 

oculated  with  virulent  cultures  of  the  anthrax  bacillus  septi- 
cemia  is  produced.  Large  numbers  of  the  bacilli  are  found  in 
the  blood,  and  may  be  crowded  together  in  the  capillaries  of  the 
liver  and  kidneys.  Men  are  occasionally  affected,  especially 
those  whose  occupation  brings  them  in  contact  with  cattle  or 
with  the  hides  and  wool  of  animals  that  die  of  the  disease.  The 
infection  usually  occurs  through  wounds  of  the  skin,  where  it 
produces  a  localized  inflammation  known  as  malignant  pustule. 
Anthrax  of  the  lungs  or  "wool-sorter's"  disease  may  be  acquired 


FIG.  83. — Bacillus  of  anthrax.     Stick-culture  in  gelatin. — (Gunther.) 

by  inhalation  of  material  containing  the  spores  of  the  bacilli. 
Infection  by  way  of  the  intestine  occurs  occasionally  but  is  less 
common.  Laboratory  workers  engaged  in  studying  the 
anthrax  bacillus  have  been  accidentally  infected  in  a  number  of 
instances. 

The  anthrax  bacillus,  owing  to  its  large  size,  was  the  first 
of  the  pathogenic  bacteria  to  be  recognized,  and  its  study  has 
furnished  the  basis  for  much  of  our  knowledge  concerning  the 
infectious  diseases.  It  was  for  anthrax  that  Pasteur  developed 
the  idea  of  making  a  protective  vaccine,  shortly  after  he  had 


PATHOGENIC    BACTERIA.  335 

produced  a  similar  vaccine  for  chicken  cholera.  There  is 
considerable  danger  to  the  inoculated  animals  attending  the 
use  of  anthrax  vaccines.  There  is  always  more  or  less  loss 
of  the  animals  from  anthrax  vaccination  itself  and  danger  of 
spreading  the  disease. 

In  order  to  obtain  material  free  from  spores  the  blood  of 
an  animal  which  has  recently  died  of  anthrax  is  taken,  because 
anthrax  spores  do  not  form  in  the  living  body.  Cultures  made 
in  bouillon  are  kept  at  a  temperature  of  from  42°  to  43°  C. 


/ 


FIG.  84. — Anthrax  bacilli  with  square  or  slightly  concave  ends  sometimes 
seen;  fuchsin  stain.     (X  1000.) 

At  this  temperature  the  virulence  of  the  anthrax  bacillus  be- 
comes gradually  diminished.  In  time  the  virulence  is  so  far 
diminished  that  rabbits  will  survive  inoculation,  and  even- 
tually also  mice  and  guinea-pigs,  which  are  extremely  suscep- 
tible to  anthrax.  Small  doses  of  a  culture  of  extremely  weak 
virulence  are  given  to  the  animals  which  it  is  desired  to  pro- 
tect, like  cattle  and  sheep,  and  subsequently  a  somewhat  more 
virulent  culture  is  employed.*  The  method  is  neyer  used  in 
human  beings. 

*  For  details  as  to  the  results  of  this  method  see  V.  A.  Moore.     Infectious 
Diseases  of  Animals.     1906. 


336  MANUAL    OF    BACTERIOLOGY. 

Bacillus  Influenzas. — A  small  bacillus,  0.2  to  0.3  /"by  0.5  /*, 
with  rounded  ends.  It  does  not  form  spores,  is  not  motile 
and  is  decolorized  by  Gram's  method.  .  It  is  aerobic,  grows 
only  in  the  incubator,  and  upon  media  containing  hemoglobin. 
The  medium  is  prepared  by  smearing  sterile  blood  over  the 
surface  of  a  tube  of  agar.  Fresh,  uncoagulated  blood  may, 
with  care,  be  mixed  with  melted  agar  sufficiently  cooled;  the 
mixture  may  be  poured  into  tubes  and  slanted;  the  tubes  should 


•" 


FIG.  85. — Anthrax  bacilli  in  the  capillaries  of  the  liver  of  a  mouse,  sketched 
from  a  section  stained  with  fuchsin. 


be  tested  in  the  incubator  before  using.  The  blood  of  some 
animals,  as  the  pigeon  and  rabbit,  may  be  used  instead  of 
human  blood.*  The  colonies  are  small  and  transparent,  look- 
ing like  little  drops  of  water,  not  becoming  confluent. 

Of  a  large  number  of  bacilli,  the  majority  are  destroyed  in 
twenty-four  hours  or  less  by  drying.  They  die  out  in  a  similar 
manner  in  water.  Experiments  upon  animals  up  to  this  time 
are  not  conclusive.  For  diagnostic  purposes,  the  sputum 

*Centralblatt  fur  Bakteriologie.     Bd.  XXXII.     Original,     p.  267. 


PATHOGENIC    BACTERIA.  337 

should  be  carefully  collected  in  a  sterile  bottle.  If  the  particles 
of  sputum  are  likely  to  have  become  contaminated,  rinse  in 
sterile  water.  Inoculate  on  ordinary  agar  and  on  blood- agar. 
The  influenza  bacillus  should  grow  only  on  the  blood- agar  and 
have  the  other  characters  above  mentioned.  Any  organism 
that  grows  on  both  the  ordinary  and  the  blood-agar  must  be 
rejected.  As  far  as  is  known,  this  organism  attacks  spon- 
taneously only  human  beings.  It  probably  does  not  grow  out- 
side the  body  in  nature.  In  cases  of  influenza  it  is  found  in 
the  mucous  discharges  and  in  the  bronchi  and  longs.  It  is 
the  predominating  organism  in  some  cases  of  bronchitis.* 
According  to  Canon,  the  bacilli  may  sometimes  be  found  in  the 
blood.  Wollsteint  found  the  influenza  bacillus  in  the  throats 
and  nasal  secretion  in  a  number  of  cases  in  children  suffering 
from  other  diseases  than  influenza,  but  failed  to  find  it  in  the 
normal  children.  She  concludes  that  the  organism  is  present  only 
in  cases  where  the  air  passages  are  affected.  In  cases  where  it 
has  been  found  in  apparently  healthy  individuals  there  should 
be  a  careful  inquiry  into  the  previous  history,  since  the  in- 
fluenzea  bacillus  may  persist  for  a  long  time.  Nevertheless, 
apparently  healthy  individuals  may  be  carriers  of  the  organism. 
She  further  concludes  that  there  is  no  justification  for  the  term 
pseudoinfluenza  bacillus,  and  regards  the  organisms  described 
by  others  as  such,  to  be  merely  variations  of  the  same  organism. 
Pertussis  Bacillus. — WollsteinJ  isolated  an  organism 
from  the  sputum  of  cases  of  pertussis  which  resembled  the 
influenza  bacillus  in  its  refusal  to  grow  upon  any  but  hemo- 
globin containing  media.  Morphologically  it  was  slightly 
larger  than  the  influenza  bacillus.  Wollstein  resorted  to  the 
Kitasato  method  of  washing  the  sputum  in  several  changes  of 
sterile  water  before  plating.  This  was  done  on  agar  to  which 

*See  Lord.     Boston  Medical  and  Surgical  Journal     December  8,  1902. 
•\Journ.  Exp.  Med.     Vol.  VIII.     1906.     pp.  681-691. 
Uourn.  Exper.  Med.     Vol.  VII.     1905.     pp.  335-342- 


338  MANUAL   OF   BACTERIOLOGY. 

placental  blood  was  added.  She  describes  the  organisms  as 
short,  plump  ovoid  cells.  Gram,  negative.  Colonies  on  agar 
transparent,  dew-drop-like,  surrounded  each  by  a  hemolytic 
zone. 

Bacillus  Diphtherias  (Klebs-Loffler). — A  straight  or  slightly 
curved  bacillus,  usually  1.2  to  2.5  M  in  length,  with  rounded  or 
slightly  pointed  ends,  remarkable  for  showing  irregularities 
of J^ form,  sometimes  being  club-shaped  or  spindle-shaped; 
branching  forms  have  been  found.*  It  is  not  motile  and  does 
not  form  spores. 

There  are  two  principal  forms:  One  short  and  relatively 
plump  which  takes  a  very  intense  uniform  stain;  the  other,  that 
presented  in  the  accompanying  photograph,  Fig.  81,  shows  the 
irregularities  mentioned.  This  irregular  form  shows  sharply 
marked,  intensely  stained  portions  alternating  with  clear  un- 
stained bands  running  across.  This  alternation  of  stained  and 
unstained  areas  is  often  quite  symmetrical  in  the  rods,  giving 
them  the  appearance  of  being  striped  at  almost  regular  inter- 
vals. At  other  times  the  stained  portion  is  at  one  or  both  of 
the  swollen,  club-shaped  ends.  With  methylene-blue  the 
stained  portions  often  appear  distinctly  red.  Considerable 
practice  is  necessary  to  acquire  the  familiarity  required  to  make 
diphtheria  diagnoses. 

It  is  best  stained  with  watery  solutions  of  the  aniline  dyes, 
especially  Loffler's  alkaline  methylene-blue.  Very  character- 
istic pictures  are  obtained  by  the  method  of  Neisser: 

SOLUTION  No.  i. 

Methylene-blue,  i 

Alcohol  (96  per  cent.),  20 

Distilled  water,  950 

Glacial  acetic  acid,  50 

SOLUTION  No.  2. 

Bismarck  brown,  i 

Boiling  distilled  water,  500 

*Hill.     Journal  Medical  Research.     Vol.  VII.     1902. 


PATHOGENIC    BACTERIA. 


339 


Stain  the  cover-glass  preparation  which  has  been  fixed  in  the 
flame  in  No.  i  one  to  three  seconds;  wash  in  water;  stain  in  No. 
2  three  to  five  seconds;  wash  in  water;  mount  as  usual.  The 
body  of  the  bacillus  is  stained  pale  brown,  with  dark  blue  spots, 
especially  at  the  ends  (Fig.  87).  In  regard  to  the  Gram  scain, 
some  strains  retain  the  stain  while  others  do  not.  Hamilton 
and  Horton*  found  that  of  18  cultures  isolated  by  them,  n 
were  Gram  negative,  7  were  Gram  positive. 


W 


FIG.  86. — Bacillus  of  diphtheria.     (X  1000.) 

The  diphtheria  bacillus  is  peculiar  in  staining  irregularly; 
certain  spots  stain  more  sharply  than  other  portions,  and 
darkly  stained  spots  are  likely  to  occur  at  the  ends.  It  is  a 
facultative  anaerobe.  It  grows  most  rapidly  in  the  incubator, 
and  slowly,  or  not  at  all,  below  20°  C.  Gelatin  is  not  liquefied. 
It  may  be  cultivated  on  various  alkaline  culture-media,  but 
grows  best  on  Loffler's  blood-serum  mixture  (page  75).  On  this 

*Journ.  Infect.  Dis.     Vol.  III.     1906.     p.  132. 


340 


MANUAL    OF    BACTERIOLOGY. 


medium  the  growth  consists  of  small  white  or  cream-colored, 
slightly  elevated  colonies,  which  may  become  confluent.  The 
morphology  of  the  bacillus  is  most  characteristic  when  it  is  culti- 
vated on  blood-serum.  It  also  grows  upon  glycerin-agar. 
On  potato  it  grows  only  if  the  potato  is  first  treated  with  soda 
solution  (i  per  cent.)  so  as  to  neutralize  the  acidity  of  this 
medium.*  In  alkaline  bouillon  containing  dextrose  or  muscle- 


FIG.  87. — Bacillus  of  diphtheria  stained  by  FIG.  88. — Swab  and  culture- 

Neisser's  method.     (X  1000.)  tube  used  in   the  diagnosis  of 

diphtheria. 

sugar  the  reaction  becomes  acid  in  forty-eight  hours. t  The 
reaction  of  the  bouillon  subsequently  becomes  alkaline.  The 
growth  may  form  a  pellicle  over  the  surface  of  the  bouillon.  It 
has  also  been  successfully  cultivated  on  various  media  to  which 
egg-albumen  has  been  added. 

It  is  killed  by  a  moist  heat  at  about  60°  C.  in  ten  minutes. 
It  is  very  resistant  to  drying. 

*  Giinther.     Loc.  cit. 

f  Kolle  and  Wassermann.     Loc.  cit.     p.  769-770. 


PATHOGENIC    BACTERIA. 


341 


Bacteriological  Diagnosis  oj  Diphtheria. — In  many  large 
cities  the  bacteriological  diagnosis  of  diphtheria  is  undertaken 
by  boards  of  health.  The  methods  used  differ  somewhat  in 
detail,  but  are  similar  in  the 
main,  and  are  based  upon  the 
procedure  devised  by  Biggs  and 
Park  for  the  Board  of  Health  of 
New  York  City.  Two  tubes  are 
furnished  in  a  box.  The  tubes 
are  like  ordinary  test-tubes, 
about  three  inches  in  length, 
rather  heavy  and  without  a 
flange.  Both  are  plugged  with 
cotton.  One  contains  slanted 
and  sterilized  Loffler's  blood- 
serum  mixture  (Fig.  88);  the 
other  contains  a  steel  rod, 
around  the  lower  end  of  which  a 
pledget  of  absorbent  cotton  has 
been  wound.  These  tubes  con- 
taining the  swabs  are  sterilized, 
and  it  would  seem  that  an  effi- 
cacious method  would  be  to 
sterilize  them  first  in  the  auto- 
clave and  subsequently  in  the 
dry  sterilizer.  The  swab  is  wiped 
over  the  suspected  region  in  the 
thoat,  taking  care  that  it  touches 
nothing  else,  and  is  then  rubbed 
over  the  surface  of  the  blood- 
serum  mixture.  The  swab  is  re- 
turned to  its  test-tube  and  the  cotton  plugs  are  returned  to  their 
respective  tubes.  The  plugs,  of  course,  are  held  in  the  fingers 
during  the  operation,  and  care  must  be  taken  that  the  portion 


FIG.  '89. — Bacillus   of   diphtheria, 
culture   on   glycerinagar. 


342  MANUAL   OF    BACTERIOLOGY. 

of  the  plug  that  goes  into  the  tube  touches  neither  the  finger  nor 
any  other  object.  The  principles,  in  fact,  are  the  same  as 
those  laid  down  in  general  for  the  inoculation  of  culture-tubes 
with  bacteria  (see  page  78).  In  board  of  health  work  these 
tubes  are  returned  to  the  office.  When  it  is  desirable,  a  second 
tube  may  be  inoculated  from  the  swab.  The  tubes  are  placed 
in  the  incubator,  where  they  remain  for  from  twelve  to  15  hours 
and  a  microscopic  examination  is  then  made  of  smear  prep- 
arations stained  with  Loffier's  methylene-blue.  After  use  the 
tubes  and  swabs  should  be  most  carefully  and  thoroughly 
sterilized. 

On  Loffler's  blood-serum  kept  in  the  incubator  the  bacil- 
lus of  diphtheria  grows  more  rapidly  than  the  other  organ- 
isms which  are  ordinarily  encountered  in  the  throat,  a 
property  which  to  a  certain  extent  sifts  it  out,  as  it  were, 
from  them,  and  makes  its  recognition  with  the  microscope 
easy  in  most  cases.  The  appearance  of  the  bacilli  under 
the  microscope  is  quite  characteristic.  Colonies  of  strepto- 
cocci frequently  look  very  like  those  of  the  bacillus  of  diph- 
theria but  those  two  are  easily  distinguished  from  each  other 
with  the  microscope.  The  diagnosis  of  the  diphtheria  bacillus 
in  practice  is  made  from  the  character  of  the  growth  upon  the 
blood-serum  and  the  microscopical  examination,  taking  into 
account  the  size  and  shape  of  the  bacilli,  with  the  frequent  oc- 
currence of  irregular  forms  and  the  peculiar  irregularities  in 
staining,  and  this  usually  suffices;  but  in  doubtful  cases  a  sec- 
ond culture  should  be  made  from  the  throat,  and  if  necessary 
corroborates  by  the  inoculation  of  a  guinea-pig. 

The  very  large  number  of  examinations  that  have  been  made 
by  various  boards  of  health  have  shown  that  the  diphtheria 
bacillus  may  persist  in  the  throat  for  a  long  time — occasionally 
several  weeks  after  the  patient  has  apparently  recovered;  also 
that  diphtheria  bacilli  are  occasionally  found  in  the  throat 
when  there  is  an  inflammatory  condition  without  any  pesudo- 


PATHOGENIC    BACTERIA.  343 

membrane,  and  that  they  not  only  appear  in  an  apparent 
healthy  throat,  especially  in  hospital  nurses  and  in  children 
who  have  been  associated  with  cases  of  diphtheria,  but 
also  in  those  who  have  had  no  tracable  contact  with  diph- 
theria cases.*  It  has  been  found  that  bacilli  sometimes 
occur  in  the  throat  which  have  all  thenmorphological  and  cul- 
tural properties  of  the  diphtheria  bacillus,  but  which  are  de- 
void of  virulence  when  tested  upon  animals.  Such  diphtheria 
bacilli  have  frequently  been  called  pseudodiphtheria  bacilli.  A 
bacillus  closely  resembling  the  diphtheria  bacillus,  but  without 
virulence,  has  been  found  in  xerosis  of  the  conjunctiva.  It  is 
called  the  xerosis  bacillus.  If  not  a  transformed  diphtheria 
bacillus,  it  is  at  least  closely  related.  The  diphtheria  bacillus 
is  subject  to  wide  variations  in  morphology,  so  that,  in  dealing 
with  unknown  cultures  where  the  forms  are  not  characteristic 
and  injection  into  animals  is  without  result,  it  may  be  difficult 
to  decide  whether  or  not  the  organisms  are  diphtheria  bacilli. 
Consequently  another  view  with  regard  to  pseudodiphtheria 
bacilli  has  arisen.  While  recognizing  that  non-virulent  diph- 
theria bacilli  occur,  it  is  also  claimed  that  a  distinct  pseudodiph- 
theria bacillus  exists,  different  from  the  diphtheria  bacillus, 
though  resembling  it.  It  is  shorter,  stains  more  evenly,  shows 
no  polar  granules  by  Neisser's  method  of  staining,  does  not 
produce  acid  in  dextrose-bouillon,  and  is  not  pathogenic  to 
animals  and  does  not  produce  diphtheria  toxin.  It  is  found 
occasionally  in  the  nose  and  throat  and  has  no  connection  with 
diphtheria,  according  to  this  view.f  But  there  are  some  who 
hold  that  there  is  no  pseudodiphtheria  bacillus,  and  that  the 

*Sholiy.     Journ.  Inject.  Dis.     Vol.  IV.     1907.     pp.  337-346. 

f  The  different  sides  of  this  question  will  be  found  fully  discussed  by  the 
following:  Wesbrook,  Wilson  and  McDaniel.  Transactions  of  the  Association 
American  Physicians.  1900.  Gorham.  Journal  Medical  Research.  Vol.  VI. 
1900.  A.  Williams.  Ibid.  Vol.  VIII.  1902.  Denny.  Ibid.  Vol.  IX. 
1903.  Alice  Hamilton.  Journal  of  Infectious  Diseases.  Vol.  I.,  No.  4.  1904. 
Graham  Smith.  Journal  of  Hygiene.  Vol.  IV.  1904. 


344  MANUAL   OF    BACTERIOLOGY. 

organism  so  called  it  merely  a  more  or  less  modified  form  of  the 
diphtheria  bacillus. 

At  a  meeting  of  the  Association  of  American  Pathologists  and  Bacteriol- 
ogists, Perkins*  described  two  organisms,  or  one  organism  with  variations  of 
characteristics,  which  he  came  across  in  routine  examinations  of  cultures  from 
throats  of  persons  suspected  of  having  diphtheria,  and  which  he  regards  as 
likely  to  lead  to  error.  The  points  of  difference  from  the  diphtheria  which 
are  given  below  would  seem,  however,  to  make  a  differentiation  not  very  difficult 
with  proper  care.  Although  resembling  the  bacillus  of  diphtheria  bacillus,  it 
differs  in  having  a  greater  regularity  in  outline  than  the  latter,  and  in  fact  that 
this  regularity  persists  after  treatment  with  acetic  acid.  The  chromophylic 
granules  take  a  deeper  stain  than  in  the  diphtheria  bacillus.  The  organism 
is  motile  and  forms  spores.  In  old  cultures  there  is  distinct  thread  formation, 
with  true,  but  inconstant  branching  in  cultures  over  a  month  old.  Gelatine  is 
slowly  liquefied.  To  guard  against  error  it  would  seem  necessary  for  those 
who  make  routine  examinations  for  diphtheria  merely  to  test  the  organisms 
which  resemble  diphtheria  bacilli  for  motility  in  order  to  avoid  the  possibility 
of  mistaking  this  organism  of  Perkins  for  the  genuine  diphtheria  bacillus. 
The  formation  of  spores  after  twenty-four  hours  would  also  enable  one  to 
distinguish  the  Perkins  organism  from  the  true  diphtheria  bacillus. 

Persons  who  harbor  the  diphtheria  bacilli  in  their  throats 
whether  they  show  any  clinical  symptoms  or  no  should  be  re- 
garded as  a  menace  to  those  around.  Their  throats  should  be 
actively  treated  with  suitable  antiseptics  and  water,  and  fre- 
quently examined  for  the  presence  of  the  organism.  It  is 
probable  that  in  this  way  such  persons  can  be  quickly  rendered 
harmless  to  those  about  them. 

The  diphtheria  bacillus  is  pathogenic  for  guinea-pigs,  rabbits, 
cats,  chickens,  pigeons  and  to  a  somewhat  lesser  extent,  for 
dogs,  goats,  cattle  and  horses. f  When  it  is  injected  into  them 
it  produces  a  toxemia.  In  the  guinea-pig,  which  is  especially 
susceptible,  local  inflammation  results,  and  death  occurs 
usually  in  two  or  three  days.  The  bacilli  are  found  to  be  con- 
fined to  the  vicinity  of  the  wound,  and  not  usually  to  be  dis- 

*Personal  communication  to  Dr.  Williams. 

fPark.  Pathogenic  Bacteria  and  Protozoa.  New  York  and  Philadelphia. 
1905.  p.  192. 


PATHOGENIC    BACTERIA.  345 

seminated  throughout  the  whole  body.  The  death  of  the 
animal,  therefore,  is  due  to  the  poisons  elaborated  by  the 
diphtheria  bacilli — either  poisons  introduced  at  the  original 
injection,  or  substances  produced  by  the  bacilli  which  may 
have  multiplied  in  the  animal's  body.  The  internal  viscera, 
especially  the  liver,  often  exhibit  small  areas  consisting  of 
necrotic  cells;  a  transudation  of  serum  takes  place  in  the  great 
serous  cavities,  and  the  lymph-nodes  are  swollen.  A  genuine 
diphtheritic  membrane  may  be  produced  on  the  trachea  of  a 
young  kitten  by  rubbing  into  it  a  part  of  a  culture  of  the  diph- 
theria bacillus. 

As  is  well  known,  the  pseudomembranous  affection  pro- 
duced by  the  diphtheria  bacillus  in  man  is  generally  seen  in 
the  larynx  and  pharynx.  Membranous  rhinitis  is  also  caused 
by  the  diphtheria  bacillus.  On  the  other  hand,  pseudomem- 
branous affections  of  the  larynx  and  pharynx  indistinguishable 
from  diphteria  except  by  bacteriological  examination  may  be 
produced  by  streptococci.*  Pseudomembranes  occurring  in  the 
throat  during  scarlet  fever  and  measles  may  be  due  to  the  diph- 
theria bacillus,  but  are  more  often  caused  by  streptococci.  The 
affection  known  as  membranous  croup  is  usually  diphtheria  of 
the  larynx  produced  by  the  diphtheria  bacillus.  The  diph- 
theria bacillus  is  a  rare  cause  of  puerperal  fever.  Although 
the  uninjured  skin  is  not  attacked  by  the  diphtheria  bacillus, 
it  may  be  present  in  pseudomembranes  on  wounded  surfaces, . 
usually  in  connection  with  diphtheria  in  the  throat.  Most 
pseudomembranes  formed  upon  wounds  of  the  skin  are  pro- 
duced by  other  bacteria  than  the  diphtheria  bacillus,  as  is  also 
the  case  with  the  pseudomembranous  inflammations  of  the 
intestines  and  bladder.  Although  such  inflammations  are 
often  called  "diphtheric,"  it  must  be  remembered  that  the  ex- 

*Bissell.  Medical  News.  May  31,  1902.  American  Journal  Medical 
Sciences.  February,  1903.  Review  of  Work  of  Massachusetts  Boards  of 
Health. 


346  MANUAL    OF    BACTERIOLOGY. 

pression  is  used  in  an  anatomical  sense,  meaning  that  a  fibrin- 
ous  pseudomembrane  has  formed,  extending  deeply  into  the 
tissues,  which  is  not  necessarily  caused  by  the  diphtheria 
bacillus. 

The  diphtheria  bacillus  is  usually  found  associated  with 
other  pathogenic  bacteria  in  cases  of  diphtheria.  The  pus 
cocci  and  the  pneumococcus  very  frequently  complicate  the 
disease.  It  seems  that  the  diphtheria  bacillus  may  be  either 
the  cause  of  the  primary  disease  and  prepare  the  way  for  sec- 
ondary invasion,  or  on  the  other  hand  it  may  follow  or  ac- 
company a  primary  infection  of  some  other  organism.  In 
other  words,  cases  starting  as  diphtheria  may,  and  usually  do 
become  complicated  by  pus  coccus  invasion,  or  cases  starting 
pus  coccus  infection  or  as  measles. or  scarlet  fever  or  pneu- 
monia may  be  complicated  by  a  secondary  infection  with  the 
diphtheria  bacillus 

In  cases  of  diphtheria  in  man,*  the  diphtheria  bacillus  is 
generally  found  limited  to  the  vicinity  of  the  pseudomembrane, 
and  at  autopsies  it  is  not  usually  found  in  the  internal  viscera, 
excepting  in  the  lungs,  where  diphtheria  bacilli  may  or  may  not 
be  present  when  diphtheria  is  complicated  with  bronchopneu- 
monia.  The  general  symptoms  of  the  disease,  including  the 
paralysis  which  sometimes  follows  it,  are  due  to  the  toxins 
produced  by  the  bacilli  in  the  throat. 

Diphtheria  Antitoxin.^ — It  is  necessary  first  to  obtain  the  toxin  produced  by 
diphtheria  bacilli  in  a  concentrated  form.  For  this  purpose  virulent  diphtheria 
baccilli  are  cultivated  in  alkaline,  sugar-free  bouillon,  in  flasks  plugged  with 
cotton,  exposing  a  large  surface  to  the  air.  Park  %  finds  that  the  presence  of 
muscle-sugar  makes  no  difference,  provided  the  broth  is  made  sufficiently 
alkaline  to  neutralize  the  acid  formed  by  the  fermentation  of  the  sugar,  and 

*  For  a  full  study  of  the  lesions  of  diphtheria  see  the  Monograph  of  Council- 
man, Mallory  and  Pearce.  Boston.  1901. 

fSee  articles  by  Park,  A.  Williams,  Atkinson  and  T.  Smith.  Journal  of 
Experimental  Medicine.  Vol.  I.,  p.  164;  Vol.  III.,  p.  513;  Vol.  IV.,  pp.  373 
and  649.  Journal  Medical  Research.  Vol.  IX.,  p.  173. 

J  Park.     Loc.  cit.     p.  194. 


PATHOGENIC   BACTERIA.  347 

also  where  the  culture  employed  is  a  vigorous  one.*  On  the  other  hand  Theo- 
bold  Smith  f  finds  that  the  alkalinity  of  the  broth  is  not  the  essential  point  in 
the  production  of  strong  toxin,  but  that  it  is  necessary  to  use  broth  which  has 
been  fermented  with  the  colon  bacillus  to  rid  it  of  muscle-sugar.  Smith's 
directions  are  as  follows : 

Beef  infusion  prepared  in  the  usual  way  by  using  500  grams  of  finely  chopped, 
lean  beef  is  kept  in  the  cold  for  12-24  hours;  the  juice  then  expressed;  the 
reaction  brought  to  1.5  or  2  per  cent,  acid  with  normal  sodium  carbonate  solu- 
tion; heated  to  40°  C.;  inoculated  with  30-40  c.c.  of  a  24-hour  bouillon  culture 
of  B.  coli;  and  placed  in  the  high-temperature  incubator  for  16  hours  or  over 
night;  it  is  then  clarified  by  adding  the  white  of  one  egg  to  every  liter  of  infu- 
sion and  boiling  for  45-60  minutes  in  the  Arnold  sterilizer;  cooled  down  and 
filtered;  2  per  cent,  of  Witte's  peptone  and  0.5  per  cent,  of  common  salt  are 
added  and  dissolved  by  gentle  heat;  the  acidity  is  reduced  to  0.8  per  cent,  with 
sodium  carbonate;  o.i  per  cent,  of  dextrose  is  added;  steamed  for  or  boiled 
for  20-30  minutes  and  filtered;  distributed  into  Fern  bach  flasks  in  shallow 
layers,  2.5  ccm.  deep  and  sterilized  in  the  autoclave  at  110-115°  C.  for  30 
minutes.  The  cultures  of  diphtheria  which  are  employed  should  form  mem- 
branes over  the  medium  promptly,  leaving  a  clear  fluid  beneath.  Previously 
grown  beef-broth  cultures  in  test-tubes  are  used  to  inoculate  the  Fern- 
back  flasks.  The  cultures  in  these  .flasks  become  distinctly  alkaline  to 
phenolphthalein  in  6-8  days  at  which  time  the  maximum  of  toxin  has 
been  formed.  After  having  been  tested  for  purity  with  the  microscope  and  by 
cultures  they  are  treated  as  follows:  "Rendered  sterile  by  the  addition  of  10 
per  cent,  of  a  5  per  cent,  solution  of  carbolic  acid.  After  48  hours  the  dead 
bacilli  have  settled  on  the  bottom  of  the  jar  and  the  clear  fluid  above  is 
syphoned  off  or  it  is  filtered  through  ordinary  sterile  filter  paper  and  stored  in 
full  bottles  in  a  cold  place  until  needed."  The  filtrate  contains  the  toxin. 
The  toxin  is  injected  into  the  animal  from  which  the  antitoxin  is  to  be  obtained 
in  small  doses.  The  dose  depends  on  the  strength  of  the  toxin.  The  aimal 
usually  employed  is  the  horse,  which  should  be  healthy;  the  presence  of  tuber- 
culosis and  glanders  should  have  been  excluded  by  testing  with  tuberculin 
and  mallein;  the  possible  presence  of  tetanus  should  also  be  considered  (see 
page  330). 

The  injection  is  repeated  at  intervals  of  about  one  week,  using  larger  and 
larger  doses,  until  the  animal  is  able  to  tolerate  a  very  large  dose  indeed — as 
much  as  300  c.c.,  or  even  more.  If  the  treatment  is  successful,  the  general 
condition  of  the  animal  should  not  suffer.  The  injections  last  over  a  long 
period — usually  about  two  or  three  months.  The  general  condition  of  the 
animal  remaining  good,  the  toleration  of  these  large  doses  of  toxin  is  presumed 
to  indicate  the  existence  of  a  concentrated  antitoxic  substance  in  the  blood. 

*Park.     Loc.  cit.     p.  205-206. 

"\Journ.  Exper.  Med.     Vol.  IV.     1899.     pp.  273-297. 


348  MANUAL    OF    BACTERIOLOGY. 

Small  quantities  of  blood  may  be  withdrawn  from  time  to  time,  and  the  serum 
tested  for  its  antitoxic  strength.  When  a  satisfactory  serum  has  been  attained, 
the  animal  may  be  bled  and  the  serum  saved  for  therapeutic  purposes. 
Through  an  incision  in  the  skin  a  trocar  is  inserted  into  the  jugular  vein.  The 
blood  is  drawn  into  sterilized  flasks  with  every  precaution  to  insure  sterility. 
The  blood  is  allowed  to  coagulate  and  is  placed  for  a  time  in  the  ice -chest. 
The  serum  is  then  withdrawn  with  sterilized  pipettes.  Small  amounts  of 
chemical  germicides,  as  carbolic  acid  or  chloroform,  are  sometimes  added  to 
assist  in  preserving  it.  This  serum  is  the  so-called  antitoxin  used  in  medical 
practice. 

Many  methods  have  been  recommended  for  concentrating  antitoxin.  Gibson* 
recommends  a  modification  of  the  method  devised  by  Pick.  Briefly  stating 
and  omitting  many  details,  Pick's  method  consists  of  precipitating  the  anti- 
toxic serum  with  a  saturated  solution  of  c.p.  ammonium  sulphate.  Gibson 
redissolves  this  precipitate  with  a  saturated  solution  of  sodium  chloride,  repre- 
cipitates  with  ammonium  sulphate  or  better  with  dilute  acetic  acid  and  dializes 
in  running  water  over  night,  neutralizes  the  acetic  acid  if  this  has  been  used, 
and  dializes  in  running  water  for  two  days  longer.  This  preparation  is  said 
to  possess  many  advantages.  For  one  thing  there  is  said  to  be  no  rash  fol 
lowing  its  use  as  with  so  many  preparations  of  antitoxin. 

Since  antitoxin  is  not  obtained  as  a  pure  chemical  substance,  and  conse- 
quently cannot  be  weighed  and  measured  as  other  theraputic  preparations, 
an  arbitrary  standard  to  express  the  potency  of  the  serum,  called  an  immunity 
unit,  has  been  devised  by  Behring  and  modified  by  Ehrlich.f  Formerly  this 
unit  was  taken  to  be  10  times  that  amount  of  antitoxic  serum  which  just  neu- 
tralized 10  fatal  doses  of  toxin  for  guinea-pigs  weighing  250  grams.  In  other 
words,  the  exact  amount  of  a  certain  toxin  required  to  kill  a  guinea-pig  weigh- 
ing 250  grams  in  four  days  having  been  determined  by  inoculating  a  number 
of  guinea-pig?.,  ten  times  this  amount  was  put  into  each  of  a  number  of  test- 
tubes,  and  the  antitoxin  to  be  tested  was  added,  a  slightly  different  amount  to 
each  tube  of  toxin.  The  contents  of  each  tube  was  then  injected  into  a  sepa- 
rate guinea-pig. 

If  any  of  the  animals  survived,  the  amounts  of  antitoxin  in  the  tubes  with 
which  they  had  been  inoculated  having  been  noted,  the  smallest  of  these  amounts 
— i.  e.,  the  smallest  amount  found  necessary  to  neutralize  the  toxin — was  re- 
gared  as  one-tenth  of  an  antitoxic  unii.  It  was  naturally  assumed  that  10 
times  this  amount  of  antitoxin  would  neutralize  100  fatal  doses.  This,  how- 
ever, was  found  not  to  be  the  case  (see  Immunity,  page  223).  So  the  revised 
standard  now  employed  in  Germany,  France,  America  and  other  countries  is 
the  unit  recommended  by  Ehrlich.  This  consists  of  comparing  the  antitoxin 

*Journ.  Biol.  Chem.  Vol.  I.  Jan.,  1908.  pp.  161-170.  Abstr.  in  Bun. 
de  I' lust.  Past.  Vol.  IV.,  No.  9.  1906.  p.  426. 

fRosenau.  Immunity  Unit  for  Diphtheria  Antitoxin.  Public  Health  and 
Marine  Hospital  Service.  Bulletin  21.  1905. 


PATHOGENIC    BACTERIA. 


349 


to  be  standardized  with  antitoxin  specially  prepared  by  Ehrlich  for  the  pur- 
pose. This  antitoxin  of  Ehrlich  is  supplied  to  the  various  public  and  private 
institutions  where  antitoxin  is  prepared,  and  is  carefully  standardized  against 
very  fresh  toxin,  which  therefore  contains  little  toxoid. 

The  Ehrlich  standard  antitoxin  is  really  used  in  the  first  place  to  determine 
the  strength  of  a  given  toxin,  which  in  turn  is  used  to  determine  the  value  of 
antitoxin  to  be  standardized.  The  actual  method  is  to  mix  varying  amounts 
of  the  toxin  to  be  tested  each  with  one  unit  of  the  standard  antitoxin,  and  that 
mixture  which  just  suffices  as  proved  by  experiment  to  kill  a  25o-gram  guinea- 
pig  in  three  or  four  days  is  designated  L  +  (see  Immunity,  page  224);  the 
mixture  which  is  just  neutral  is  called  LO.  That  amount  of  antitoxin  which 
ju^t  neutralizes  L  +  contains  one  antitoxic  unit  according  to  this  method  of 
standardizing. 

The  injection  of  guinea-pigs  with  antitoxin  serves  the  double,  purpose  of 
determining  the  potency  of  the  antitoxin  and  also  of  determining  the  presence 
or  absence  of  pathogenic  substances,  such  as  tetanus  toxin. 

It  has  been  found  possible  to  prepare  antitoxin  of  a  high 
degree  of  concentration,  so  that  500  to  1500  units  may  be  con- 
tained in  a  quantity  of  serum  which  it  is  practicable  to  give  at  a 
single  hypodermic  injection.  The  large  volume  of  statistics 
that  have  been  collected  from  hospitals  and  from  physicians  in 
private  practice  indicates  that  the  use  of  diphtheria  antitoxin 
has  effected  a  very  great  reduction  in  the  mortality  from 
diphtheria.  Aside  from  the  curative  value  of  diphtheria  anti- 
toxin, it  is  of  value  as  a  preventative  when  administered  to 
persons  who  are  exposed.  Where  a  case  of  diphtheria  occurs 
in  a  family  all  the  other  members  beside  the  patient  should  be 
given  an  immunizing  dose  of  antitoxin — about  500  units 
suffices.  When  this  is  done,  the  disease  is  limited  to  the  one 
case. 

Bacillus  Fusiformis.* — Under  this  name  an  organism  was 
isolated  by  Vincent  and  claimed  by  him  to  be  the  cause  of 
certain  infectious  pseudomembranous  ulcerations.  Vincent's 
observations  have  been  more  or  less  corroborated  by  others, 
but  the  crucial  tests  of  cultivation  and  inoculation  do  not  seem 

*  Vincent.     Ann.  del'  Institute  Pasteur.     1896.     p.  488. 


350  MANUAL   OF    BACTERIOLOGY. 

to  have  been  made  successfully  to  prove  the  pathological  sig- 
nificance of  the  organism. 

The  bacillus  is  described  as  non-motile,  varying  from  6-8  /* 
to  10-20  /*  in  length.  It  appears  mostly  or  exclusively  to  be 
mixed  with  other  bacteria.  Bernheim  observed  the  organism 
in  ulcerative  stomatitis  mixed  with  spirochetae. 

Bacillus  Tuberculosis. — A  slim  bacillus  with  rounded  ends 
1.5  to  4  /*  in  length.  It  very  frequently  presents  a  beaded  ap- 
pearance, owing  to  its  being  dotted  with  bright,  shining  spots. 


FIG.  90.— Bacillus  tuberculosis,  from  a  pure  culture.     (X  1000.) 

Branching  forms  have  been  described.  The  tubercle  bacillus 
is  considered  by  some  to  be  a  member  of  the  actinomyces  group. 
It  is  not  motile.  It  has  not  been  proved  that  spores  are  formed  ; 
nevertheless  certain  structures,  like  caseous  lymph-nodes,  have 
been  shown  to  be  capable  of  infecting  guinea-pigs  with  tuber- 
culosis, although  tubercle  bacilli  could  not  be  demonstrated 
in  them  with  the  microscope.  This  makes  it  seem  possible 
that  the  organisms  were  present  as  spores  which  eluded  the 
microscopical  examination.  The  tubercle  bacilli  stain  with  the 


\**'f  *^ 

*\A  ^ 

J^/         > 


PATHOGENIC    BACTERIA.  351 

ordinary  aniline  dyes  and  by  Gram's  method,  but  they  do  not 

take  the  stains  as  readily  as  most  other  bacteria,  and  require 

somewhat  longer  exposure  to  the  dye  than  other  bacteria,  on 

warming  of  the  stain.     When  once  stained,  however,  with 

aniline-water  dyes  or  carbol-fuchsin,  they  are  not  readily  de- 

colorized by  acids  and  alcohol,  which  fact  distinguishes  them 

from  all  other  known  bacteria  excepting  the  leprosy  bacillus, 

the  smegma  bacillus,  possibly  the  bacillus  of  syphilis  (Lust- 

garten),  and  certain  bacilli  found  in  milk,  butter  and  cow-dung 

and  on  various  grasses.     All  of  these  may  resist  decolorization 

by  acids  or  alcohol,  and  some  resist 

both.     They  must  always  be  kept  in 

mind  in  making  a  diagnosis  of  tuber-  '        m± 

culosis.     (See  pages  33  and  36.)     In 

examining   sputum   it  is  particularly       ^ 

important  to  bear  in  mind  that  acid- 

proof     bacilli,     resembling     tubercle 

bacilli,  have  been  found  in  rare  cases 

in  gangrene  of   the    lung.      But   the        FIG.  91.—  Branching  form 

of  tubercle  bacillus  from  a 

organisms   found   in   these   cases  are     culture.    (X  1000.) 
longer  than  tubercle  bacilli,  as  a  rule, 

and  branch  more  often,  besides  being  less  resistant  to  decolor  • 
ization.*  The  tubercle  bacilli  appear  to  owe  their  peculiar 
staining  properties  to  fatty  substances  contained  in  the  bodies 
of  the  bacilli.  In  stained  preparations  the  bacillus  usually 
appears  very  distinctly  beaded,  owing  to  the  presence  of 
stained  areas  which  alternate  with  unstained  areas;  these 
unstained  areas  have  been  considered  by  some  to  be  spores. 

The  Bacillus  tuberculosis  is  aerobic.  It  requires  certain 
special  media  for  its  cultivation  (see  below),  and  it  does  not 
grow  so  readily  when  it  is  first  inoculated  from  tuberculous 
material  from  man  or  lower  animals  as  it  does  subsequently. 

Ophuls.  Journal  Medical  Research.  Vol.  III.  1902.  Ohlmacher. 
Journal  A  merican  Medical  A  ssociation.  1901. 


352  MANUAL   OF    BACTERIOLOGY. 

But  after  it  becomes  accustomed  to  the  artificial  environment, 
it  may  be  readily  cultivated  on  a  number  of  different  media, 
though  its  growth  is  always  relatively  slow  as  compared  with 
that  of  many  other  bacteria.  It  does  not  grow  at  a  tempera- 
ture below  29°  C.,  and  the  best  temperature  is  around  38°  C.  It 
cannot,  therefore,  be  cultivated  upon  gelatin  even  if  this  were  a 
suitable  medium  otherwise.  It  grows  well  upon  blood-serum, 
where  the  growth  becomes  visible  in  from  ten  to  fourteen  days  in 


.-*. 


• 


FIG.  92. — Bacillus  tuberculosis  in  sputum,  stained  with  carbol-fuchsin  and 
methylene-blue.     Photomicrograph  in  two  colors.     (X  1000.) 

the  incubator.  It  forms  a  dry,  mealy,  scaly  mass,  elevated  above 
the  surface,  of  a  grayish-brown  color.  It  also  grows  upon 
glycerin- agar;  or  glycerin-bouillon,  on  which  it  forms  a  pel- 
licle; upon  potato;  upon  milk  containing  i  per  cent,  of  agar 
and  upon  coagulated  egg  (see  page  76).  It  is  important  to 
have  the  medium  moist.  It  can  be  cultivated  from  tuberculous 
sputum  only  with  great  difficulty.  It  is  best  to  obtain  it  from 
the  tissues  of  an  animal  that  has  died  of  tuberculosis,  where  the 


PATHOGENIC    BACTERIA.  353 

tubercle  bacilli  may  be  found  unmixed  with  other  bacteria. 
Pieces  of  tissue  should  be  taken  with  the  precautions  necessary 
to  avoid  contamination,  and  should  be  broken  up  and  rubbed 
over  the  surface  of  the  medium.  The  tubes  must  be  closed 
with  sealing-wax,  paraffin  or  rubber  stoppers,  or  covered  with 
rubber  caps,  to  prevent  drying  in  the  incubator.  If  rubber 
caps  are  used,  they  should  first  be  left  in  i-iooo  bichloride  of 
mercury  for  an  hour,  and  the  cotton  plug  should  be  burned 
before  putting  on  the  rubber  cap.  A  number  of  tubes  should 
be  inoculated,  using  rather  large  particles  of  the  tuberculous 
material.  Among  the  tubes  inoculated,  some  are  apt  to  show 
no  growth.  After  the  organism  has  once  been  grown  upon 
a  culture-medium  it  may  be  propagated  with  less  difficulty. 

The  statement  in  regard  to  the  action  of  germicidal  agents  in 
general  may  be  repeated  here  with  regard  to  their  action  upon 
the  tubercle  bacillus.  The  results  of  laboratory  experiments 
to  determine  the  effects  of  the  various  agents  upon  the  tubercle 
bacillus  cannot  with  safety  be  absolutely  relied  upon  in  practice 
to  destroy  the  bacilli.  The  measures  employed  in  practice 
should  in  all  cases  be  much  more  drastic  than  would  be 
indicated  as  just  sufficient  by  the  results  of  laboratory  experi- 
ments. Absolute  destruction  by  fire  should  be  resorted  to 
whenever  this  is  feasible,  and  next  to  this  sterilization  in  the 
autoclave  at  115  to  120°  C.  Chemical  disinfectants  are 
uncertain,  particularly  in  the  disinfection  of  sputum,  feces 
and  the  like.  The  following  statements  in  regard  to  the 
effects  of  various  germicides  are,  therefore,  of  more  theoretical 
than  of  practical  interest. 

The  bacilli  are  very  resistant  to  drying.  They  remain  alive 
for  about  two  months  when  kept  dry.  They  are  also  more 
resistant  to  the  destructive  action  of  heat  when  dry  than  when 
moist.  They  have  been  known  to  remain  alive  after  one  hour's 
heating  at  100°  C.  when  dry;  but  when  they  are  exposed  in 
water,  milk,  beef-broth  or  other  fluids  they  are  killed  at  55°  C. 

23 


354  MANUAL    OF    BACTERIOLOGY. 

in  four  hours,  at  60°  C.  in  30  minutes,  at  65°  C.  in  15  minutes, 
at  70°  C.  in  10  minutes,  at  80°  C.  in  five  minutes,  at  95°  C.  in 
one  minute.  These  temperatures  are  of  importance  in  the 
matter  of  the  sterilization  of  milk,  since  it  is  important  that 
milk  should  be  heated  as  little  as  necessary.  Theobald 
Smith  showed  that  the  scum  which  forms  on  the  surface  of 
milk  when  it  is  heated  protects  the  tubercle  bacilli  whkh  are 
in  the  scum  from  the  effects  of  the  heat.  Sunlight  destroys 
the  bacilli  quickly,  but  in  practice  the  protective  coating  of 
sputum  or  other  material  may  interfere  with  the  action  of  the 
sunlight.  In  fact  the  piotection  afforded  by  the  sputum  may 
operate  in  all  cases  where  the  bacilli  are  sought  to  be  destroyed. 
It  is  not  destroyed  always  by  the  gastric  juice  as  is  seen  in  those 
cases  referred  to  below  in  which  the  bacilli  are  found  in  the 
feces  of  persons  affected  with  phthisis,  and  who  swallow  their 
sputum.  It  has  furthermore  been  shown  by  direct  experi- 
ment, as  well  as  by  the  occasional  occurrence  of  primary 
intestinal  tuberculosis,  however  rare,  this  may  be  compara- 
tively speaking.  In  fact  the  view  that  the  bacilli  are  not 
destroyed  by  the  gastric  juice  is  the  basis  for  Behring's  con- 
tention that  all  forms  of  tuberculosis  are  acquired  by  ingestion 
of  the  bacilli.  They  are  destroyed  in  sputum  by  carbolic  acid 
in  the  proportion  of  equal  parts  of  a  five  per  cent,  solution  of 
the  carbolic  acid  to  the  amount  of  sputum.  The  fumes  from 
4  pounds  of  burning  sulphur  to  each  1000  cubic  feet  of  air 
space  kills  the  tubercle  bacillus  in  8  hours  provided  the 
bacilli  are  freely  exposed  and  the  atmosphere  is  kept  moist.* 
Formaldehyde  is  quicker  but  not  more  efficient  in  the  propor- 
tion of  10  ounces  of  formalin  to  1000  cubic  feet  of  space. f 

It  is  not  known  to  grow,  except  in  artificial  cultures,  outside 
of  the  animal  body.  It  is  the  cause  of  tuberculosis  in  man. 
It  produces  tuberculosis  in  apes,  cows,  hogs,  sheep,  horses, 

*Park.     Loc.  cit.     p.  200. 

t/wa. 


PATHOGENIC    BACTERIA. 


355 


rabbits,  guinea-pigs,  cats,  field-mice  and  occasionally  in  other 
animals.  Guinea-pigs  and  rabbits  are  extremely  susceptible. 
A  guinea-pig  inoculated  with  tuberculous  sputum  (provided 
it  does  not  die  of  septicemia,  due  to  the  pyogenic  micrococci 
which  are  frequently  present  in  sputum)  will  present  a  swelling 
of  the  neighboring  lymph-nodes  in  the  course  of  two  to  four 
weeks,  and  will  die  as  a  rule  in  from  four  to  eight  weeks, 
although  the  time  may  be  longer. 

Tuberculosis  in  cattle  (German,  Perlsuchf)  is  characterized  by  large,  nodular 
lesions,  with  a  marked  tendency  to  become  fibrous,  caseous  and  calcified.  The 
tubercle  bacilli  of  cattle  differ  somewhat  from  those  of  human  tuberculosis,  as 
was  noted  by  Theobald  Smith.*  Whether  or  not  men  could  be  infected  with 
bovine  tubercle  bacilli  has  been  a  question  that  has  been  warmly  debated  in 
recent  years.  There  seems  no  longer  room  for  doubt  that  such  infection 
does  take  place;  also  that  cattle  may  be  infected  with  human  tubercle  bacilli. 
Bovine  tubercle  bacilli  are  more  virulent  for  some  animals,  as  rabbits,  than 
human  tubercle  bacilli.f 

The  tubercle  bacillus  is  present  in  all  forms  of  tuberculosis, 
in  the  sputum  from  the  lungs  of  persons  suffering  with  phthisis, 
in  all  tissues  affected  with  tuberculosis,  as  in  the  skin  in  lupus; 
in  tuberculous  glands  in  all  situations,  in  the  cervical  glands 
in  scrofula,  in  the  mesenteric  glands  in  intestinal  tuberculosis; 
it  is  present  also  in  the  feces  in  intestinal  tuberculosis,  in  the 
urine  in  tuberculosis  of  the  urinary  apparatus.  Wherever 
there  is  a  tuberculous  lesion  in  any  location  in  man  or  in  the 
lower  animals,  and  in  the  excreta  and  in  the  secretions  from 
tuberculous  organs  the  tubercle  bacillus  is  to  be  found.  Not 
only  in  such  locations  but  also  in  certain  conditions  where 
there  is  no  lesion.  It  has  been  found  in  the  mouth,  throat 
and  nose  of  persons  who  show  no  symptoms  or  signs  of  tuber- 
culosis, but  who  associate  intimately  with  tuberculous  persons. 
It  is  found  in  the  glands  of  persons  dying  from  other  causes 

* Journal  Experimental  Medicine.     Vol.  III.,  p.  451- 

fTheobald  Smith.  Medical  News.  February  22,  1902.  Various  bulletins 
from  the  Bureau  of  Animal  Industry.  Adami.  Philadelphia  Medical  Journal. 
February  22,  1902.  Ravenel.  University  of  Pennsylvania  Medical  Bulletin. 
May,  1902.  Lartigau.  Journal  Medical  Research.  Vol.  VI.  1901.  Wolbach 
and"  Ernst.  Ibid.  XII.,  p.  295.  Theobald  Smith.  Ibid.  XIII.,  p.  299. 


356  MANUAL    OF    BACTERIOLOGY. 

than  tuberculosis  in  a  very  large  proportion  of  cases.  Indeed, 
the  statement  is  made  by  competent  authorities  that  nearly 
all  persons  above  the  age  of  T8  years  probably  have  latent 
tuberculous  foci  in  their  bodies.* 

In  nearly  every  case  which  comes  to  autopsy,  whether  the 
subject  showed  evidence  of  tuberculosis  during  life  or  no, 
tubercle  bacilli  may  be  found  in  the  lymph-glands,  if  not 
directly  with  the  microscope  at  least  by  the  inoculation  of 
susceptible  animals  with  bit  of  the  gland.  In  this  way  as 
many  as  90  per  cent,  and  over  of  autopsies  in  hospitals  have 
revealed  the  presence  of  tubercle  bacilli  whether  there  were 
lesions  of  tuberculosis  or  no  lesions  of  tuberculosis.  The 
feces  of  persons  who  are  suffering  from  pulmonary  tuberculosis 
may  contain  the  bacilli  even  in  cases  in  which  there  is  no 
involvement  of  the  intestine.  In  such  cases  the  bacilli  are 
swallowed  with  the  sputum,  and  are  discharged  from  the 
intestines. 

In  external  nature  the  tubercle  bacillus  is  found  for  the  most 
part  in  the  air  surrounding  tuberculous  patients.  They  are 
found  not  only  in  the  dry  air  attached  to  floating  particles, 
.but  Fliigge  has  shown  that  they  are  thrown  into  the  air  by 
tuberculous  persons  in  every  act  of  coughing,  and  that  they 
remain  for  a  long  time  floating  about  on  the  little  globules  of 
sputum. 

The  lesion  produced  by  the  tubercle  bacilli  in  the  tissues 
of  men  and  the  lower  animals-  is  called  a  tubercle,  which  in 
the  beginning  is  a  grayish-white  area  about  the  size  of  a 
millet-seed.  In  sections  of  the  tissue  young  tubercles  are 
found  to  present  several  different  structures.  Near  the 
center,  one  or  more  very  large  cells  called  giant-cells  occur. 
They  contain  several  or  many  nuclei  which  are  frequently 
arranged  in  a  crescentic  manner  at  one  side  of  the  cell.  Tuber- 
cle bacilli  can  sometimes  be  demonstrated  inside  of  the  giant- 

*Giinther.     Loc.  cit.     p.  472. 


PATHOGENIC    BACTERIA.  357 

cell.  Except  possibly  in  the  very  youngest  tubercles,  a  small 
area  of  necrotic  tissue  will  usually  be  found  at  the  center  of  the 
tubercle. 

Around  the  giant-cells  and  the  necrotic  area  are  seen  large 
cells  with  distinct  nuclei  which  resemble  epithelial  cells,  and  are 
often  called  epithelioid  cells;  they  are  also  often  termed  granu- 
lation cells,  and  represent  an  attempt  at  the  formation  of 
granulation  tissue.  But  no  new-formed  blood-vessels,  such  as 
are  found  in  granulation  tissue  as  a  rule,  occur  in  the  tubercle. 
Tubercle  bacilli  may  also  be  found  among  the  epithelioid  cells. 
Outside  of  these  epithelioid  cells  is  another  layer  of  small  cells 
called  lymphoid  cells,  which  represent  leukocytes  that  have 
appeared  in  this  situation  as  a  part  of  the  inflammatory  reaction 
excited  by  the  presence  of  the  tubercles.  The  zone  of  lymph- 
oid cells  may  be  very  indistinct  or  wanting.  Frequently  it 
may  be  very  difficult  to  make  out  that  the  cells  are  arranged  in 
distinct  zones  at  all,  for  instead  of  distinct  tubercles,  the  tuber- 
cle bacillus  may  produce  a  diffuse  form  of  inflammatory  tissue. 
The  cells  are  imbedded  in  a  matrix  consisting  of  the  connective 
tissue  originally  belonging  to  the  part,  to  which  some  fibrin 
may  be  added.  In  addition  to  the  fact  that  no  new  blood- 
vessels are  formed  to  maintain  the  nutrition  of  these  newly 
formed  cells,  the  small  vessels  included  in  the  tubercle  and 
around  it  suffer  from  inflammatory  changes.  Owing  to  these 
causes  and  to  a  toxic  substance  formed  by  or  in  the  tubercle 
bacilli,  degenerative  changes  and  necrosis  take  place  at  the 
central  part  of  the  tubercle.  As  a  result  of  these  degenerative 
changes  the  center  of  the  tubercle  becomes  converted  into  a 
dry,  yellowish-white,  friable  mass,  resembling  dry  cream- 
cheese.  Such  material  is  said  to  be  caseous,  and  the  process 
is  called  caseation.  Prudden  and  Hodenpyl  found  that  the 
injection  of  dead  tubercle  bacilli  into  animals  produced  lesions 
having  the  histological  characters  of  tubercles,  but  caseation 
did  not  take  place. 


358  MANUAL    OF    BACTERIOLOGY. 

The  small  tubercles  first  formed  are  called  gray  or  miliary 
tubercles.  As  they  become  larger  they  also  frequently  become 
confluent.  The  larger,  confluent,  caseous  tubercles  are  often 
called  yellow  tubercles.  Swollen  tuberculous  lymph  nodes  of 
the  neck  are  among  the  manifestations  of  the  condition 
formerly  known  as  scrofula. 

Masses  of  caseous  tubercles  sometimes  undergo  softening. 
In  the  lungs  the  discharge  of  the  softened  material  results  in 
the  formation  of  a  cavity.  This  formation  of  a  cavity  in  the 
lungs  is  frequently,  if  not  usually,  accompanied  by  secondary 
infection  with  pyogenic  micrococci.  Caseous  tuberculous 
masses  may  become  partly  calcified.  Very  often  they  may  be 
encapsulated  by  new  formed  fibrous  or  scar  tissue.  It  is 
possible  for  tuberculosis  to  become  cured  for  all  practical 
purposes  by  means  of  this  process.  Autopsies  on  human 
subjects  have  shown  that  such  cures  not  rarely  take  place, 
especially  in  tuberculosis  of  the  lungs  occurring  over  a  localized 
area.  The  statistics  of  autopsies  vary  widely  as  to  the  number 
of  persons  that  at  some  time  of  life  suffer  from  tuberculosis, 
from  25  or  30  per  cent,  up  to  much  higher  figures.  When  a 
tuberculous  area  has  become  caseous  and  encapsulated  and 
apparently  quiescent,  it  is  possible  for  it  to  be  excited  to 
renewed  activity  under  suitable  conditions,  and,  owing  to  the 
softening  and  the  discharge  of  infected  material  into  one  of  the 
vessels  or  cavities  of  the  body,  a  wide-spreading  and  rapidly 
fatal  tuberculosis  may  follow. 

Tuberculosis  may  become  disseminated  throughout  the 
body  from  a  small  focus  as  a  starting-point.  The  tubercle 
bacilli  may  travel  through  the  lymph-spaces  and  affect 
adjacent  tissues,  some  of  them  reaching  the  nearest  group  of 
lymph-nodes.  In  tuberculosis  of  the  lungs  it  is  usual  also  to 
find  tubercles  in  the  bronchial  lymph-nodes,  and  in  tuber- 
culosis of  the  intestines  there  is  also  tuberculosis  of  the  mesen- 
teric  lymph-nodes.  The  disease  may  travel  along  the  serous 

\ 


PATHOGENIC    BACTERIA.  359 

surfaces  and  become  widely  scattered  throughout  a  cavity  like 
that  of  the  pleura  or  peritoneum.  The  bacilli  may  be  expelled 
on  some  mucous  surface  and  be  carried  along  it  to  infect  some 
point  farther  on,  as  happens  when  the  larynx  becomes  infected 
in  tuberculosis  of  the  lung,  and  when  in  the  same  disease 
tuberculous  sputum  is  swallowed  and  leads  to  infection  of  the 
intestines.  Finally,  the  infectious  material  may  enter  the 
blood-vessels,  especially  the  veins,  and  be  swept  along  with 
the  blood-current  to  become  scattered  generally  throughout 
the  body.  In  such  cases  we  are  likely  to  have  general  or  acute 
miliary  tuberculosis.  Almost  every  organ  of  the  human  body 
may  be  infected  by  tuberculosis.  Among  the  most  common 
may  be  mentioned  the  lungs,  the  lymph-nodes,  the  bones,  the 
intestines,  the  skin,  the  meninges,  and  the  serous  membranes. 

Harbitz*  found  as  a  result  of  his  observations  that  primary 
tuberculosis  of  the  lymph  glands  is  quite  frequent  in  adults, 
not  only  in  the  thorax,  but  also  in  the  abdomen,  and  especially 
in  the  cervical  nodes,  often  it  is  generally  distributed  in  the 
lymphatic  system  having  extended  through  years  and  tens  of 
years. 

Infection,  as  far  as  we  know,  is  always  to  be  attributed 
directly  or  indirectly  to  some  preexisting  case  of  tuberculosis 
in  man  or  the  lower  animals. 

The  mode  of  entry  into  the  body  of  both  man  and  animals 
is  a  matter  of  liveliest  dispute;  some  holding  with  Fliigge  that 
the  commonest  mode  of  entrance  is  by  way  of  inhalation 
into  the  lungs;  others  maintaining  with  Behring  that  the 
entrance  is  always  by  the  alimentary  tract,  f  Those  who  hold 
the  latter  view  have  this  much  to  present  in  evidence  that,  as 
Raven  el  showed,  the  tubercle  bacillus  may  pass  through  the 

*Journ.  Infect.  Dis.    Vol.  II.     1905.     pp.  143-247. 

fVarious  bulletins  of  the  Department  of  Agriculture.  Revenel.  Journal 
of  Medical  Research.  X.,  p.  460.  Ibid.  Transactions  of  the  American  Public 
Health  Association.  XXIX.  Calmette  and  Guerin.  Annales  de  I' Institute 
Pasteur.  III.,  p.  1024,  IV.,  p.  636. 


360  MANUAL    OF    BACTERIOLOGY. 

intestines  to  the  lungs  without  leaving  any  trace  of  their  passage. 
The  others  hold  that  where  animals  are  fed  with  tuberculous 
material,  particles  are  insufflated  in  their  passage  down  the 
esophagus.  The  fact  that  cases  have  been  found  in  which  the 
tonsils  were  the  only  seat  of  tuberculosis  in  the  whole  body 
shows  that  in  such  cases  at  least  the  infection  was  from  the 
air  passing  over  the  tonsils. 

The  dissemination  of  the  tubercle  bacillus  is  doubtless 
very  largely  due  to  the  prevalent  habit  of  expectorating  in 
public  places.  Out  of  fifty-six  samples  of  sputum  collected 
in  street  cars  by  Dr.  W.  G.  Bissell,  City  Bacteriologist  in 
Buffalo,  four  were  tuberculous.  In  forty-eight  samples  taken 
from  the  floors  of  the  public  building  by  Dr.  C.  R.  Orr,  of  the 
pathological  laboratory  of  the  University  of  Buffalo,  tubercle 
bacilli  were  found  three  times.  According  to  the  researches 
of  Nuttall,  a  person  suffering  from  tuberculosis  may  expec- 
torate many  millions  of  tubercle  bacilli  in  the  course  of  twenty- 
four  hours.  Coughing  and  sneezing  may  serve  to  dissemi- 
nate the  bacilli  (see  page  175). 

Concerning  the  occurrence  of  tubercle  bacilli  in  cow's  milk 
and  butter,  and  in  beef,  see  pages  151,  351  and  152. 

Cases  have  been  recorded  in  which  the  disease  was  trans- 
mitted from  the  mother  to  the  child  in  the  uterus;  how  fre- 
quently this  happens  is  uncertain.  It  is  usual  to  attribute 
greater  importance  to  an  inherited  tendency  to  tuberculosis 
than  to  the  inheritance  of  the  tubercle  bacilli  themselves.* 

Agglutination  of  the  tubercle  bacillus  is  said  to  occur  with 
the  serum  of  cases  of  tuberculosis  under  certain  circumstances. 
The  reaction  does  not  seem  likely  to  be  of  practical  value. 

Tuberculin  is  made  by  concentrating  a  culture  of  tubercle 
bacilli  grown  in  glycerin-bouillon  to  one-tenth  of  its  original 
volume,  over  a  water-bath,  and  filtering  through  a  sterilized 

*On  tuberculosis  of  the  placenta  see  Warthin.  Journ.  Infect.  Dis.  Vol.  IV. 
1907.  pp.  347-368. 


PATHOGENIC    BACTERIA.  361 

Pasteur-Chamberland  bougie  of  unglazed  porcelain.  It  there- 
fore represents  the  products  of  tubercle  bacilli.  It  was  pro- 
posed by  Koch  as  a  remedy  for  tuberculosis,  but  it  has  not 
met  with  great  success,  and  is  little  used  as  a  therapeutic 
agent.  It  has  been  found,  however,  of  great  value  in  the 
diagnosis  of  tuberculosis,  especially  in  cattle.  When  tuberculin 
is  injected  into  a  tuberculous  animal  there  results  considerable 
general  disturbance,  of  which  the  mo'st  noticeable  evidence 
is  a  sudden  rise  in  temperature,  while  hyperemia  is  excited 
around  the  tuberculous  area.  In  a  healthy  subject  the  in- 
jection produces  no  reaction;  but  there  is,  nevertheless,  danger 
attending  its  use.  To  avoid  the  objections  to  the  injection  of 
tuberculin  in  human  beings,  Calmette*  and  Wolfe-Eisner 
independently  recommended  the  application  of  tuberculin  to 
the  conjunctiva  as  a  means  of  diagnosis.  A  marked  hyper- 
aemia  of  the  conjunctiva  follows  this  procedure  in  tuberculous 
individuals,  while  this  is  very  slight  or  entirely  absent  in 
healthy  persons.  In  tuberculous  patients  aside  from  some 
discomfort  and  interference  with  vision  there  is  no  serious 
consequence  as  a  rule,  though  in  some  cases  there  is,  however, 
considerable  oedema  and  even  purulent  exudation.  As  a 
diagnostic  measure  in  cattlef  it  has  been  found  accurate  in 
the  great  majority  of  cases.  Concerning  tuberculosis  in  cows, 
see  page  151.  Supposing  that  some  curative  principle  exists 
in  the  bodies  of  the  tubercle  bacilli  themselves  which  could 
not  be  procured  from  cultures  deprived  of  their  bacilli  by 
nitration  through  porcelain,  Koch  has  recently  proposed  a  new 
form  of  tuberculin  called  "tuberculin  R,"  which  consists  of  an 
extract  made  from  dried  and  pulverized  living  tubercle  bacilli. 
The  value  of  this  new  tuberculin  as  a  remedy  is  at  least  doubt- 
ful, and  physicians  are  disposed  to  regard  it  as  dangerous. 

*Calmette.     Comptes  rendus  de  1' Academic  des  Sciences.     June  17,  1907. 
fFor  details  as  to  its  use  in  cattle  see  V.  A.  Moore.     Infectious  Diseases  of 
Animals.     1906.     p.  196. 


362  MANUAL    OF    BACTERIOLOGY. 

Weber*  obtained  a  positive  reaction  in  five  healthy  physi- 
cians who  tried  the  intraocular  injection.  The  explanation 
in  these  cases  is  to  be  found  in  the  fact  that  the  men  experi- 
mented upon  were  in  the  habit  of  sitting  up  reading  late  by 
artificial  light. 

Immunity  from  tuberculosis  has  been  attained  experimentally  to  a  certain 
degree.  In  very  old  cultures  the  virulence  of  tubercle  bacilli  sometimes  becomes 
greatly  diminished.  Animals  which  survive  injections  of  such  bacilli  may 
afterward  withstand  large  doses  of  virulent  bacilli. f 

Friedmannt  has  succeeded  in  rendering  guinea-pigs  and  also  cattle  immune 
to  injection  with  virulent  tubercle  bacilli  by  injections  of  cultures  of  turtle 
tubercle  bacilli.  A  single  injection  of  such  cultures  which  are  in  themselves 
apparently  harmless,  confers  a  high  degree  of  immunity  upon  cattle. 

Acid-proof  bacilli  resembling  tubercle  bacilli  have  been  alluded  to  a  number 
of  times  (pages  33,  36,  163,  351).  A  number  of  such  bacilli  have  been  culti- 
vated, such  as  those  of  butter  and  grass.  Injected  into  animals  they  may  pro- 
duce nodules  more  or  less  like  tubercles.  In  these  nodules  they  sometimes 
assume  forms  resembling  the  fungus  of  actinomycosis.  The  tubercle  bacillus 
rarely  shows  similar  forms.  All  the  bacilli  of  this  class,  including  the  tubercle 
bacillus,  sometimes  show  branching.  It  is  probable  that  the  bacilli  of  this 
group  are  related  to  the  fungus  of  actinomycosis.  §  Similar  organisms  have 
been  found  in  fishes,  in  whom  they  produce  nodules  resembling  tubercles;  it 
is  quite  possible  that  the  latter  organisms  are  tubercle  bacilli,  which  have  been 
modified  by  an  altered  environment.  Another  acid-proof  bacillus  has  been 
found  which  is  pathogenic  to  rats,  producing  lesions  of  the  skin  with  nodules; 
the  disease  appears  in  wild  rats  in  certain  localities. 

Tuberculosis  of  Birds. — Fowls,  ducks  and  other  birds  sometimes  suffer 
from  tuberculosis  due  to  a  bacillus  closely  resembling  the  tubercle  bacillus 
of  mammals.  It  has  similar  staining  properties.  It  sometimes  grows  in  long, 
branching  forms.  It  differs  somewhat  from  the  tubercle  bacillus  of  mammals 
in  its  cultural  properties.  The  liver  is  the  organ  most  often  affected.  Guinea- 
pigs  are  much  less  susceptible  to  it  than  to  mammalian  tuberculosis.  Rabbits 
are  somewhat  susceptible,  though  less  so  than  to  mammalian  tuberculosis. 

Pseudotuberculosis. — Guinea-pigs  and  other  rodents  sometimes  present 
lesions  macroscopically  very  similar  to  those'  of  tuberculosis,  in  which,  however, 
the  tubercle  bacilli  cannot  be  found.  These  affections  appear  not  to  be  tuber- 
culosis at  all,  and  their  nature  is  not  well  understood.  Several  organisms 
have  been  found  in  them,  all  of  which  are  entirely  unlike  the  tubercle  bacillus. 

Bacillus  Leprae  (bacillus  of  leprosy). — A  slim  bacillus  about 
4  j«  in  length.  It  is  probably  not  motile.  It  is  uncertain 

^British  Med.  Journ.  Feb.  1908.  Cited  in  Journ.  ^Amer.  Med.  Assn. 
March  i,  1908. 

fTrudeau.  New  York  Medical  Journal.  July  18,  1903.  Salmon.  Phila- 
delphia Medical  Journal.  June  13,  1903. 

%Deutsche  Med.  Wochenschr.  XXX.,  No.  46.     1904. 

§Abbott  and  Gildersleeve.  University  of  Pennsylvania  Medical  Bulletin. 
June,  1902.  Borrel.  Bacilles  Tuberculeux  et  Paratuberculeux.  Bulletin  de 
V  Institute  Pasteur.  T.  II.,  p.  409  and  p.  505. 


PATHOGENIC    BACTERIA.  363 

whether  or  not  it  forms  spores.  It  stains  by  the  Gram  and  the 
Weigert  fibrin  method  but  requires  to  be  stained  for  a  longer 
time  than  most  Gram  positive  bacteria,  and  it  is  also  colored 
by  the  methods  used  for  staining  the  tubercle  bacillus.  It 
takes  the  dye,  however,  more  readily  than  the  tubercle  bacillus. 
In  stained  preparations  it  appears  very  similar  to  the  tu- 
bercle bacillus,  and  resembles  it  in  having  alternate  colored 
and  unstained  spots.  Babes  mentions  short  branching  forms 
with  inverted  pear-shaped  extremities.  Although  several  ob- 
servers have  reported  success  in  attempts  to  cultivate  the 
bacillus  of  leprosy,  their  claims  have  been  disputed.  Organ- 
isms resembling  the  diphtheria  bacillus,  two  different  kinds 
have  been  cultivated  on  artificial  media  by  Babes*  and  others, 
but  Babes  regards  these  as  merely  adventitious. 

The  leprosy  bacilli  lie  for  the  most  part  deep  in  the  skin,  but 
Babes  also  found  them  in  the  hair-follicles.  He  also  found 
them  in  the  sputum,  nasal  secretion,  in  the  conjunctival  sac, 
in  the  sperma,  urethral  secretion  and  elsewhere,  but  not  in 
the  urine;  only  in  small  numbers  in  the  mucous  membrane 
of  the  bladder,  twice  in  the  milk,  once  in  the  feces,  in  small 
numbers  in  the  pleura  and  peritoneal  fluid.  They  are  usually 
absent  from  the  blood,  found  only  once  in  12  cases.  Often 
in  the  vaginal  secretion  in  those  affected.  They  are  very 
abundant  in  the  ulcers. 

The  results  of  inoculation  into  man  and  the  lower  animals 
of  material  coming  from  cases  of  leprosy  have  been  uncertain. 
The  bacillus  of  leprosy  has  been  found  so  constantly  in  the 
tissues  of  those  having  the  disease  that  it  is  generally  admitted 
to  be  the  specific  cause.  The  skin  and  the  peripheral  nerves 
are  the  parts  most  affected,  although  other  tissues  and  the 
internal  viscera  may  be  involved.  A  granulation  tissue, 
forming  nodules  and  thickenings,  appears  in  the  affected  parts. 
The  bacilli  are  found  in  large  numbers  in  the  nodules,  partly 

*Kolle  and  Wassermann.     Erganzungsband.     1906.     p.  160. 


364  MANUAL    OF    BACTERIOLOGY. 

outside  of  the  cells,  but  mostly  within  the  cells.  Most  persons 
who  are  affected  with  leprosy  have  the  bacilli  in  the  nasal  secre- 
tion.* The  disease  must  be  communicated  directly  from  one 
individual  to  another,  for  no  explanation  can  be  given  for 
the  appearance  of  the  infection  in  any  patient,  except  by  com- 
munication with  some  other  case.  Still  transmission  by 
contact  seems  not  to  take  place  easily. 

Bacillus  Mallei  (bacillus  of  glanders). — A  slim  bacillus  with 
round  or  pointed  ends,  which  often  shows  alternate  light  and 
dark  spots  in  stained  preparations.  Branching  forms  have 
been  described.  It  is  not  motile.  It  probably  does  not  form 
spores.  It  does  not  retain  the  stain  by  Gram's  method. 
After  staining  with  the  ordinary  aniline  dyes  it  is  easily  decolor- 
ized, and  on  that  account  it  is  difficult  to  demonstrate  in  sections 
of  tissues.  It  is  facultative  anaerobic.  It  grows  at  the  room 
temperature,  but  better  in  the  incubator.  It  grows  slowly  on 
gelatin,  and  does  not  liquefy  it,  or  only  after  a  long  time.  On 
agar  it  produces  a  moist,  white  growth;  on  blood-serum,  a 
yellowish  or  brownish  growth;  blood-serum  is  not  liquefied. 
Milk  is  coagulated  slowly,  and  the  reaction  becomes  acid. 
On  potato,  the  growth  one  or  two  days  in  the  incubator  is 
translucent  amber-yellow,  later  a  reddish  brown,  while  the 
surface  of  the  potato  becomes  discolored. 

In  the  first  few  days  on  potato,  cultures  resemble  those  of  B. 
pyocyaneus  on  this  medium.t 

In  the  horse  and  ass  it  produces  the  disease  known  as 
glanders,  which  affects  the  mucous  membrane  of  the  nasal 
cavity.  When  the  skin  is  involved,  the  disease  goes  by  the 
name  of  farcy.  In  the  nose,  nodules  appear  in  the  mucous 
membrane  which"  become  necrotic/  forming  ulcers.  They 
may  become  confluent,  and  may  extend  along  the  adjacent 
surfaces  as  far  as  the  lungs.  There  is  a  profuse  discharge 

*Giinther.     Loc.  cit.     p.  509. 

tFrothingham.     Journal  of  Medical  Research.     Vol.  VI.,  p.  334. 


PATHOGENIC    BACTERIA.  365 

from  the  nose.  The  neighboring  lymph-nodes  become 
involved  and  are  swollen,  and  nodules  may  be  present  in  the 
internal  viscera.  In  the  skin  the  nodes  lying  underneath  the 
skin  are  called  farcy-buds.  Histologically  the  nodules  con- 
sist of  a  granulation  tissue,  but  they  tend  to  break  down 
rapidly,  and  the  process  in  some  respects  is  very  like  ordinary 
suppuration. 

In  addition  to  the  spontaneous  infection  of  horses  and 
asses,  cultures  are  pathogenic*  for  guinea-pigs,  European 
field-mice  and  cats;  rabbits,  sheep  and  dogs  are  less  susceptible 
or  only  slightly  so;  also  white  and  house-mice,  and  hogs; 
cattle  are  immune.  Camels  are  susceptible,  hedgehogs  also. 
Men  are  occasionally  infected,  especially  those  who  come  much 
in  contact  with  horses.  The  mucous  membranes  of  the  nasal 
cavity  may  be  the  part  involved,  or  the  skin  or  the  internal 
viscera.  In  a  number  of  instances,  workers  in  the  laboratory 
have  been  accidentally  infected. 

The  diagnosis  of  the  disease  is  best  effected  by  the  inocu- 
lation of  a  male  guinea-pig  with  the  material  from  a  case  sus- 
pected of  being  glanders,  introducing  it  into  the  peritoneal 
cavity  (methocfe^of  Straus).  Frothingham|  describes  the 
method  of  procedure  which  he  employs  as  follows : 

The  material  from  the  suspected  animal— nasal  secretion  or 
skin  lesion  or  both — is  obtained  by  using  a  swab  such  as  is 
used  to  obtain  material  for  diagnosis  in  diphtheria.  After 
it  has  been  applied  to  the  lesion  the  cotton  plegget  is  removed 
to  a  convenient  quantity — 2  or  3  c.c. — of  sterile  water,  and 
agitated.  The  water  is  then  injected  in  equal  portions  into 
the  peritoneal  cavity  of  two  guinea-pigs.  In  about  two  to 
three  days  after  an  inoculation  of  this  kind  there  appears  a 
characteristic  swelling  of  the  testicle,  indicating  the  beginning 
of  suppuration,  which  presently  takes  place;  the  animal  usually 

*The  statements  of  different  writers  differ  considerably  with  regard  to  some 
of  these  animals. 

\Journ.  Med.  Research.     Vol.  VI.     1901.     pp.  331-340. 


366  MANUAL    OF    BACTERIOLOGY. 

dies  after  two  or  more  weeks.  At  least  two  guinea-pigs  should 
be  inoculated;  and  the  test  may  sometimes  fail,  when  it  should 
be  repeated  on  other  guinea-pigs.*  The  test  may  also  fail 
on  account  of  the  death  of  the  guinea-pigs  from  peritonitis. 

Frothingham  further  points  out  that  there  is  another 
organism  beside  B.  mallei  which  produces  ulceration  of  the 
guinea-pig  testicles  which  may,  however,  be  distinguished 
from  the  latter  on  potato  cultures  by  its  white  growth.  The 
brownish-yellow  growth  of  B.  mallei  on  potato  resembles  the 
growth  of  B.  pyocyaneus  at  first,  but  the  latter  causes  in  two 
or  three  days  a  greenish  discoloration  of  the  potato.  B.  mallei 
does  not  do  this. 

Mallein  is  a  product  obtained  from  old  glycerin-bouillon 
cultures  of  Bacillus  mallei.  The  cultures  are  sterilized  in  a 
steam  sterilizer  at  100°  C.  for  several  hours,  and  are  filtered 
through  a  Pasteur-Chamberlin  cylinder  of  unglazed  porcelain. 
The  filtrate  contains  the  products  of  the  growth  of  the  Bacillus 
mallei  and  is  of  much  the  same  character  as  tuberculin. 
Injected  into  animals  suspected  of  having  glanders,  if  it  pro- 
duces a  local  and  febrile  reaction,  the  existence  of  glanders 
is  indicated.  This  reaction  is  of  use  in  the  diagnosis  of  the 
disease  in  lower  animals,  especially  in  horses,  where  it  has  been 
largely  employed,  though  it  sometimes  fails.  An  agglutination 
reaction  has  been  described  for  the  bacillus  of  glanders. 

In  regard  to  this  reaction  Moore  and  Taylorf  find  that  it 
is  easier  to  perform  and  quite  as  accurate  as  the  mallein  test. 

Actinomyces  BovisJ  (Streptothrix  actinomyces;  Ray- 
fungus  of  Actinomycosis). — The  morphology  of  this  organism 
is  quite  different  from  that  of  most  of  the  bacteria.  It  is 
sometimes  considered  to  be  a  bacterium  of  a  higher  type.  The 
organism  appears  in  the  form  of  threads  which  show  genuine 

*Frothingham.     Journal  Medical  Research.     Vol.  VI.  1901. 
"fJourn.  Infect.  Dis.     Supplement  No.  3.     1907.     pp.  85-94. 
JHektoen.     Philadelphia    Monthly    Medical    Journal.     November,     1899. 
Ewing.     Bulletin  Johns  Hopkins  Hospital.     November,  1902. 


PATHOGENIC    BACTERIA.  367 

branching.  These  threads  make  radiating,  interlacing  masses. 
Their  external  ends  are  swollen  and  bulbous  under  certain 
conditions.  Colonies  formed  in  this  manner,  seen  under 
moderate  magnification,  have  a  radiating  appearance  which 
has  given  rise  to  the  name,  ray-fungus.  The  club-shaped 
external  ends  are  readily  distinguished  and  the  growth  possesses 
a  very  distinctive  form.  This  is  the  shape  which  the  organism 
presents  as  it  grows  in  the  animal  body.  The  club-shaped 
ends  are  generally  regarded  as  a  degenerative  or  involution 


FIG.  93. — Ray-fungus  of  actinomycosis.     Fresh,  unstained  preparation 
from  a  case  of  lump-jaw  in  a  cow.     (Diagrammatic.) 

form.  Transverse  divisions  may  sometimes  be  distinguished 
upon  the  threads.  Spherical  forms  resembling  micrococci 
may  appear  which  may  possibly  be  spores.  In  some  members 
of  this  group  spores — conidia — form  in  cultures  on  the  ends 
of  the  filaments.  While  the  organism  stains  with  the  ordinary 
aniline  dyes,  by  Gram's  method  or  the  Weigert  fibrin  stain. 
It  is  absolutely  essential  that  the  anilin  oil-gentian  violet  be 
prepared  with  a  strictly  saturated  alcoholic  solution  of  gentian 
violet,  and  that  the  stain  should  be  a  few  days  old,  not  freshly 
prepared.* 

*Gunther.     Loc.  cit.     p.  780. 


368  MANUAL    OF    BACTERIOLOGY. 

The  fungus  may  be  cultivated  upon  the  usual  culture- 
media,  though  not  easily.  It  is  facultative  anaerobic.  It 
grows  both  at  ofdinary  temperatures  and  in  the  incubator. 
The  growth  is  not  rapid.  The  colonies  are  fine,  dry,  elevated, 
irregular  in  form,  becoming  opaque.  Bulbous  ends  upon  the 
threads  do  not  usually  appear  in  cultures.  The  results  of  the 
injection  of  these  cultures  into  the  lower  animals  are  as  yet 
uncertain.  Most  authors  report  failure  to  obtain  positive 
results  of  any  kind  and  no  one  has  yet  succeeded  in  producing 
typical  actinomycosis  by  inoculating  pure  cultures.* 

The  disease  produced  by  the  ray-fungus  is  called  actino- 
mycosis. It  occurs  in  cattle  chiefly,  seldom  in  swine  and 
horses,  and  occasionally  in  man.  Infection  appears  to  be 
carried  by  grain  or  particles  of  vegetable  fiber  which  penetrate 
the  tissue.  The  presence  of  such  foreign  particles  as  well  as 
the  organisms  appears  to  favor  infection.  The  infectious 
material  frequently  enters  through  the  mouth,  especially  in 
the  vicinity  of  the  teeth,  but  it  may  also  occur  through  the  skin 
or  the  mucous  membranes.  It  leads  to  the  formation  of 
inflammatory,  tumor-like  nodules,  hence  the  name  "lump- 
jaw  "  given  to  the  disease  in  cattle.  Necrosis  of  the  tissue  takes 
place  with  the  formation  of  an  abscess.  The  pus  is  peculiar 
in  containing  small  yellowish-white  particles — so-called  "  sul- 
phur granules" — which  consist  of  little  clumps  of  the  ray- 
fungus,  and  which  readily  permit  the  disease  to  be  diagnosed 
by  the  microscope.  The  material  may  be  examined  in  the 
perfectly  fresh  condition  without  any  staining.  The  jaw  or  its 
neighborhood  is  very  frequently  affected,  or  the  disease  may  be 
present  in  other  situations  about  the  head  and  neck,  and  may 
involve  the  lungs,  the  intestines  and  the  vertebrae,  ribs  and 
other  bones.  The  disease  is  usually  localized,  but  a  number  of 
areas  may  be  affected  simultaneously. 

*Kolle  and  Wassermann.     Loc.  clt.     p.  879. 


PATHOGENIC    BACTERIA.  369 

As  a  result  of  his  research,  Wright*  comes  to  different  conclusions  from  many 
of  those  who  have  studied  the  ray-fungus.  He  regards  the  organism  as  essen- 
tially anaerobic;  seldom  found  in  external  nature;  probably  present  in  the  normal 
alimentary  canal.  He  succeeded  in  obtaining  club  shapes  in  serum  cultures, 
and  in  producing  nodules  by  inoculation  of  animals.  The  bodies  called  spores 
by  other  observers  are  not  to  be  regarded  as  spores. 

According  to  Wright's  description,  the  organism  represented  by  Fig.  89  would 
have  to  be  regarded  not  as  the  true  ray-fungus  but  as  one  of  its  saprophytic 
congeners. 

Besides  the  common  actinomyces,  there  are  numerous  other  ray-fungi,  more 
or  less  closely  related,  and  whose  pathogenic  properties  are  not  fully  deter- 
mined. Generally  speaking,  they  appear  to  be  essentially  saprophytes,  which 


FIG.  94. — Actinomyces  bovis,  smear  preparation  from  a  pure  culture, 
stained  by  Gram's  method.     (X  1000.) 


occasionally  become  parasitic  and  pathogenic  under  especially  favorable  con- 
ditions. A  number  of  species  have  been  found  in  air,  dust,  etc.,  some  of  them 
chromogenic.  Wolff  and  Israel  described  an  anaerobic  species,  pathogenic 
to  man  and  animals.  Madura  disease,  Madura  foot,  or  mycetoma  is  a  disease 
occurring  in  India  (rarely  elsewhere),  affecting  one  of  the  extremities,  character 
ized  by  swellings,  nodular  deposits  and  abscesses.  Some  cases  are  certainly 
due  to  a  member  of  the  aetinomyces  group. f 

Other  branching  organisms,  some  of  them  acid-poof,  have  been  described 


*  Wright.     Journal  of  Medical  Research.     XIII.,  p.  349. 

fCompare  Wright.     Journal  Experimental  Medicine.     Vol.  III.,  p.  421. 

24 


370  MANUAL    OF    BACTERIOLOGY. 

chiefly  under  the  name  of  streptothrix.     In  man  they  have  been  found  in  a 
variety  of  suppurative  and  necrotic  lesions,  in  particular,  bronchopneumonias.* 

Bacillus  Typhosus  (Bacillus  of  Eberth). — A  bacillus  with 
rounded  ends,  varying  in  length,  sometimes  making  very  short, 
oval  forms,  sometimes  growing  out  into  long  threads.  It  is 
very  actively  motile,  and  possesses  numerous  flagella  which 
arise  from  all  parts  of  the  surface.  It  does  not  form  spores. 
It  is  not  stained  by  Gram's  method,  but  it  may  be  colored  with 


FIG.  95. — Bacillus  of  typhoid  fever.     (X  1000.) 

the  ordinary  aniline  dyes,  when  the  stain  will  frequently  be 
somewhat  irregular.  It  may  be  stained  in  sections  of  tissues 
from  cases  of  typhoid  fever,  with  the  aniline  dyes,  such  as 
Loffler's  alkaline  methylene-blue.  It  is  a  facultative  anaerobe. 
It  grows  at  ordinary  temperatures,  better  in  the  incubator,  but 
grows  rather  more  slowly  than  B.  coli  communis.  Gelatin  is 
not  liquefied.  Young  surface  colonies  in  gelatin  appear 

*Norris  and  Larkin.  Journal  Experimental  Medicine.  Vol.  V.,  p.  155. 
Musser.  Philadelphia  Medical  Journal.  September  7,  1901.  Flexner. 
Journal  Experimental  Medicine.  Vol.  III.  MacCallum.  Centralblatt  jur 
Bakteriologie.  Orginal.  Bd.  XXXI.  1902. 


PATHOGENIC    BACTERIA.  371 

whitish,  with  irregular  borders  and  more  or  less  wrinkled 
surfaces,  when  slightly  magnified.  It  grows  on  the  ordinary 
media,  and  the  growths  are  whitish.  Bouillon  is  clouded. 
Milk  becomes  slightly  acid,  but  is  not  coagulated.  In  media 
containing  dextrose,  acid  is  formed  but  no  gas.  In  lactose- 
bouillon  neither  acid  nor  gas  is  formed,  although  when  grown 


FIG.  96. — Bacillus  of  typhoid  fever,  stained  by  Loffler's  method 
to  show  flagella.     (X  1000.) 

in  milk  the  typhoid  bacilli  produce  an  acid  reaction;  but  this 
acidity  is  not  due  to  a  fermentation  of  the  milk-sugar,  but  to  a 
substance  resembling  glucose  as  was  pointed  out  by  Theobald 
Smith. 

In  Dunham's  peptone  solution  indol  is  not  formed  in  one  or 
two  days,  but  in  cultures  grown  for  10  days  at  37°  C.  there  is 
always  indol  formed,*  as  a  rule. 

On  the  lactose-litmus-gelatin  or  agar  of  Wurtz  the  blue  tinge  possessed  by 
colonies  of  the  typhoid  bacillus  on  this  medium  is  made  use  of  to  distinguish 

*Chantemesse,  Morris  (Quoted  from  Gunther  Loc.  cit.     p.  526). 


372  MANUAL    OF    BACTERIOLOGY. 

them  from  colonies  of  the  colon  bacillus  and  other  bacteria  which  form  acids 
from  lactose.  Neutral-red  has  been  used  in  the  same  manner,  as  it  is  not  altered 
by  the  typhoid  bacillus,  but  is  changed  by  the  colon  bacillus  to  a  yellow  color. 
This  medium  is  prepared  by  adding  to  neutral,  plain  agar  .05  grams  of  neutral- 
red  to  i  liter  of  agar  or  i  per  cent,  of  a  saturated  aqueous  solution  of  neutral 
red,  some  also  add  0.3  per  cent,  dextrose.  The  material  to  be  examined  is 
shaken  up  with  the  melted  agar  and  the  tubes  placed  upright  in  the  incubator 
at  35-37°  C.  These  constitute  the  "shake  tubes"  mentioned  in  books  on  water 
examination.  Neutral-red  may  also  be  used  in  the  same  proportions  as  an 
addition  to  beef-broth,  and  the  cultures  made  in  fermentation  tubes. 

On  potato  it  usually  forms  what  is  called  an  invisible  growth; 
that  is,  although  no  development  is  apparent  to  the  eye, 
numerous  bacilli  may  be  shown  under  the  microscope  in  smear 
preparations  made  from  the  surface  of  potato  inoculated 
about  forty-eight  hours  previously.  Occasionally  a  slight 
visible  growth  is  seen  on  potato. 

The  typhoid  bacillus  is  killed  at  60°  C.  in  ten  minutes  moist 
heat;  though  Clark  and  Gage  found  that  individual  bacilli 
may  withstand  80°  C.  in  fluid  cultures  for  5  minutes.  It  resists 
drying  well.  It  can  survive  in  soil  and  sewage  a  long  time.  . 

For  a  comparison  of  the  properties  of  the  typhoid  bacillus 
and  the  colon  bacillus  see  the  latter. 

There  it  probably  no  other  organism  associated  with  an 
infectious  disease  which  presents  so  much  difficulty  in  its 
identification  in  given  cases  as  the  typhoid  fever  bacillus,  and 
yet  there  is  none  which  so  frequently  demands  early  and 
prompt  identification.  Bacteriologists  are  constantly  con- 
fronted with  the  demand  from  communities,  physicians,  and 
laymen  to  give  a  positive  answer  as  to  whether  a  specimen  of 
water,  milk,  feces  or  other  material  does  or  does  not  contain 
the  typhoid  bacillus.  This  demand  has  led  to  many  efforts 
on  the  part  of  bacteriologists  to  work  out  special  methods 
for  the  isolation  and  identification  of  the  organism.  These 
efforts  have  placed  in  the  hands  of  those  who  have  had  long 
training  the  means  of  saying  with  some  probability,  but 
hardly  yet  with  absolute  certainty,  in  all  cases  at  least,  that  the 
typhoid  bacillus  is  or  is  not  present  in  the  material  examined. 


PATHOGENIC    BACTERIA.  373 

The  crucial  test  of  animal  inoculation  which  furnishes  in  the 
case  of  anthrax,  tuberculosis,  glanders  and  other  diseases 
such  a  sure  means  of  diagnosis  cannot  be  resorted  to  in  the 
case  of  typhoid  fever  for  the  reason  that  experiment  animals 
are  not  subject  to  spontaneous  typhoid  and  do  not  take  the 
disease  on  inoculation  with  cultures  of  the  typhoid  bacillus. 
It  is  true  that  inoculation  of  animals  with  the  cultures  is 
followed  by  disease,  and  the  experiments  at  one  time  rather 
encouraged  the  hope  that  the  results  obtained  by  the  inocu- 
lation of  animals  with  cultures  of  the  typhoid  bacillus  would 
furnish  a  trustworthy  means  of  diagnosis.  But  the  symptoms 
and  lesions  produced  by  the  inoculation  of  typhoid  bacilli 
are  so  similar  to  those  produced  by  the  inoculation  with  certain 
other  bacteria  that  such  inoculations  cannot  be  depended 
upon  for  differentiation.  Some  strains  of  the  very  bacteria, 
those  of  the  colon  group,  which  it  is  important  to  separate  from 
the  typhoid  bacillus  are  so  similar  to  the  typhoid  in  pathogenic 
properties  for  experiment  animals  that  a  diagnosis  between 
them  on  this  ground  is  not  possible,  and  it  becomes  necessary 
to  resort  to  cultural  characteristics  and  to  certain  other  more 
or  less  unsatisfactory  tests. 

Of  the  various  media  which  have  been  devised,  and  the 
various  tests  which  have  been  recommended  the  following 
may  be  described. 

Loffler  introduced  a  medium  consisting  of  agar-agar 
colored  with  malachite  green.  All  bacteria  including  the 
typhoid  bacillus  are  retarded  in  growth  upon  this  medium, 
and  the  typhoid  bacillus  along  with  certain  others  change  the 
color  of  the  medium  to  yellow.* 

The  medium  suggested  by  Hissf  for  the  isolation  of  the  typhoid  bacillus 
consists  of  gelatin  and  agar,  beef -extract,  sodium  chloride  and  dextrose,  and  is 
given  a  slightly  acid  reaction.  These  substances  are  used  in  different  propor- 
tions for  plate-  and  for  tube-cultures.  This  medium  is  of  a  semi-solid  character, 

*Giinther.     Loc.  cit.     p.  534. 

^Journal  Medical  Research.     Vol.  VIII.     1902. 


374  MANUAL    OF    BACTERIOLOGY. 

and  the  great  motility  of  the  typhoid  bacillus  produces  a  uniform  clouding 
of  the  medium  in  tubes,  without  gas-formation,  characters  which  distinguish  this 
organism  from  the  colon  bacillus;  in  plate -cultures  with  this  medium  the  colonies 
exhibit  peculiar  filamentous  outgrowths.  It  is  claimed  that  it  can  be  determined 
whether  organisms  are  typhoid  bacilli  or  not  after  thirty-six  hours  in  the  incu- 
bator by  this  method. 

Other  special  media  for  the  identification  of  the  typhoid  bacillus  have  been 
devised  by  Eisner,  Stoddart,  by  Capaldi  and  Proskauer,  and  by  Piorkowski.* 
The  medium  of  Stoddart  is  based  upon  principles  similar  to  those  applied  in 
the  medium  of  Hiss. 

The  Drigalsky-Conradif  method  for  isolating  the  B.  typhosus  from  water 
and  feces  is  that  now  most  employed.  The  principle  of  this  method  consists 
in  the  use  of  a  culture-medium  on  which  the  surface  colonies  of  B.  coli  and  of 
B.  typhosus  each  show  a  characteristic,  macroscopic  appearance,  so  that  they 
can  be  separated  from  one  another.  Furthermore,  the  medium  employed  is 
unfavorable  to  the  growth  of  many  bacteria  likely  to  be  present. 

The  addition  of  crystal  violet  to  the  milk-sugar-litmus-agar  inhibits  the  growth 
of  various  organisms  without  materially  affecting  the  growth  of  B.  typhosus. 
The  medium  adopted  by  Drigalsky  and  Conradi  after  many  trials  was  as 
follows : 

(a)  Three  pounds  of  chopped  beef  placed  over  night  in  2  liters  of  water. 
Strain  off,  and  boil  for  one  hour,  filter  and  add  20  grams  of  Witte's  peptone, 
20  grams  of  nutrose,  and  10  grams  of  salt.  Boil  one  hour,  filter  and  add  to  it 
60  grams  of  best  stick  agar;  boil  three  hours  over  the  flame  or  one  hour  in  the 
autoclave,  make  slightly  alkaline  to  litmus  paper,  and  boil  one-half  hour. 

(6)  260  c.c.  litmus  solution  (Kubel  and  Tiemann);  boil  for  ten  minutes; 
add  30  grams  of  c.  p.  milk-sugar,  and  boil  the  mixture  fifteen  minutes. 

(c)  Add  solution  b  to  the  hot,  melted  solution  a;  mix  thoroughly  and  correct 
the  reaction  to  weakly  alkaline  if  not  already  so. 

(d)  Add  4  c.c.  of  a  hot,  sterile  10  per  cent,  solution  of  dehydrated  soda. 

(e)  Add  20  c.c.  freshly  prepared  T^  per  cent,  solution  of  crystal  violet  (Kry- 
stallviolet  "B,"  Hochst).     This  solution  should  be  made  with  warm,  sterilized, 
distilled  water,  but  not  boiled. 

A  part  of  this  agar  is  poured  into  Petri  dishes  at  once.  The  rest  is  kept  in 
flasks,  about  200  c.c.  in  each. 

The  material  to  be  examined  is  spread  over  the  surface  of  the  plates,  not 
mixed  with  the  medium  as  is  usually  done,  the  object  being  to  obtain  surface 
colonies  only. 

The  spreading  is  done  by  means  of  a  glass  rod  12  or  14  cm.  long,  bent  at  right 
angles  about  3  cm.  from  one  end.  The  short  arm  of  the  bend  terminates 
in  a  small  knob,  and  is  dipped  into  the  material  to  be  examined  and  run  over 
the  surface  of  the  agar  in  a  series  of  the  previously  prepared  Petri  dishes. 

Drigalsky-Conradi  plates,  as  described  above,  are  made  from  water  by  using 
the  precipitate  after  centrifuging. 

Ficker  and  Hoffmann!  recommend  the  following  method  of  treating  the 

*Elsner.  Zeitschrift  fur  Hygiene.  Bd.  XXI.,  p.  25.  1895.  Stoddart. 
Journal  of  Pathology  and  Bacteriology.  Vol.  IV.,  p.  429.  1897.  Capaldi  and 
Proskauer.  Zeitschrift  fur  Hygiene,  etc.  Bd.  XXIII.,  p.  452.  1896.  Pior- 
kowski. Berliner  klinische  Wochenschrift.  p.  145.  1899. 

fDrigalsky-Conradi.  Ueber  ein  Verfahren  zum  Nachweis  der  Typhus- 
bacillen.  Zeitschrift  fur  Hygiene  und  Infectionskrankheiten.  Bd.  XXXIX. 
1902. 

|  Ficker  and  Hoffmann.  Weiteres  iiber  den  Nachweis  von  Typhusbacillen. 
Archiv  fur  Hygiene.  Bd.  XLIX.  1904. 


PATHOGENIC    BACTERIA.  375 

material  for  examination  for  the  typhoid  bacillus  before  making  the  Drigalsky- 
Conradi  plates.  They  use  an  enriching  fluid  which  has  the  property  of  in- 
hibiting the  growth  of  colon  and  other  contaminating  organisms,  while  not 
seriously  interfering  with  the  growth  of  the  typhoid  bacillus. 

This  fluid  consists  of: 

(a)  A  stock  solution  of  beef -broth.  Take  one  kilogram  of  chopped  beef; 
add  2  liters  of  distilled  water;  heat  thirty  minutes  at  50°  to  60°  C.;  stir;  boil 
for  thirty  minutes;  fill  up  water  lost  by  evaporation;  press  through  gauze; 
measure;  add  6  per  cent,  peptone  (Witte);  \  per  cent,  salt;  heat  till  peptone  dis- 
solves; filter,  distribute  into  sterilized  beer  bottles  with  patent  stoppers;  cover 
with  paper  cones;  sterilize  two  hours;  clamp  the  stoppers  to,  and  store  away. 

(fy  Enriching  fluid:  100  c.c.  of  the  above  stock  solution  measured  in  a 
sterilized  measuring  glass;  put  in  a  sterilized  Erlenmeyer  flask;  add  sodium 
hydrate  solution,  2.7  c.c.  less  than  the  quantity  required  for  phenolphthalein 
"red  point,"  as  determined  by  neutralizing  25  c.c.  Sterilize  ten  minutes — in 
steam;  allow  to  get  cool;  add  105  c.c.  of  a  1.2  per  cent,  solution  of  caffeine 
(solution  to  be  made  fresh  in  cold,  sterilized,  distilled  water  every  time).  Add 
1.4  c.c.  of  a  j1^  per  cent,  solution  of  crystal  violet;  crystal  violet  must  be  dissolved 
cold. 

(c)  Preparation  of  the  stool: 

1.  Thin  stool  allowed  to  settle,  eight-tenths  or  nine -tenths  c.c.  of  the  thin, 
upper  portion  added  to  b. 

2.  Semifluid  stool  rubbed  up  in  a  mortar  with  i  part  of  1.2  per  cent,  solution 
of  caffeine;  filter  through  sterilized  cotton-wool;  eight-tenths  or  nine-tenths  c.c. 
of  the  filtrate  added  to  b. 

3.  Thick  feces.     Rub  i  part  of  feces  with  2  parts  of  caffeine  solution,  and 
proceed  as  in  No.  2. 

In  all  three  cases  shake  thoroughly  and  place  at  37°  C. 

(d)  Search  for  typhoid  fever  bacillus.     Examine  a  hanging-drop. 

1.  If  there  are  relatively  few  bacteria,  make  6  large  Drigalsky  agar  plates: 
plate  No.  i  of  the  series  with  0.30  to  0.35  c.c.  of  the  fluid;  plate  No.  3  with  0.25 
c.c.;  plate  No.  5  with  o.io  c.c.     Plates  Nos.  2,  4  and  6  are  the  diluted  plates  from 
Nos.  i,  3. and  5. 

2.  If  there  is  abundant  growth,  7  plates  are  made:  i,  4  and  6  are  inoculated 
with  0.2  c.c.,  0.15  c.c.  and  o.i  c.c.,  respectively,  and  the  others  are  dilutions 
from  these. 

Identification  of  colonies  as  usual. 

Keep  the  rest  of  the  culture  in  the  enriching  fluid  on  ice.  If  the  first  plates 
fail  for  any  reason,  shake  this  enriching  fluid  with  glass  beads  and  make  Drigalsky 
plates  again. 

A  simpler  way  of  preparing  the  Drigalsky-Conradi  medium  is  recommended 
by  Hagemann*  as  follows: 

Liebig's  extract,  10  grams;  Witte's  peptone,  10  grams;  sodium  chloride, 
10  grams;  water,  600  c.c.  Boil  in  a  salt-water  bath  until  100  c.c.  evaporates  off. 
Add  500  c.c.  fresh,  raw,  amphoteric  milk.  Boil  and  add  agar,  10  grams.  Boil 
until  the  agar  is  nearly  dissolved;  put  in  the  autoclave  for  twenty  to  thirty 
minutes  at  110°  to  115°  C.  Filter  in  the  streaming  steam.  Divide  up  into 
sterile  Erlenmeyers,  about  200  c.c.  in  each.  Sterilize  a  short  time. 

In  using,  melt  in  a  water-bath;  add  normal  sodium  hydrate  till  the  reaction 
is  slightly  alkaline  to  litmus-paper.  Add  20  c.c.  Merck's  litmus  solution.  Also 
add  3  drops  of  a  i  per  cent,  alcoholic  solution  of  crystal  violet.  Mix  thoroughly. 
Pour  into  Petri  dishes,  and  use  as  in  the  original  method. 


*Hagemann.     Eine  Vereinfachung  des  Drigalskischen  Nahrbodens.     Hygie- 
nische  Rundschau.     Vol.  XIV.     1904. 


376 


MANUAL    OF    BACTERIOLOGY. 


M.  W.  Richardson  has  devised  an  application  of  the  serum-test  to  plate- 
colonies  suspected  of  containing  typhoid  bacilli.  If  a  typhoid  colony  be  torn 
with  a  needle,  under  moderate  magnification  "a  seething  motion  resembling 
much  the  appearance  of  a  swarm  of  bees"  may  be  seen.  This  appearance  is 
due  to  the  motility  of  the  bacteria.  If  such  a  colony  be  touched  with  a  small 
quantity  of  blood-serum  from  a  case  of  typhoid  fever,  the  motion  is  said  to  cease 
instantly  and  almost  absolutely.  Colonies  of  other  motile  bacteria  do  not 
undergo  a  corresponding  loss  of  motility. 

THE  SERUM-TEST  FOR  TYPHOID  FEVER.* 

When  a  small  quantity  of  a  culture  of  typhoid  bacilli  is  mixed  with  a  little 
blood-serum  derived  from  a  case  of  typhoid  fever,  within  a  few  minutes  the 
motility  of  the  typhoid  bacilli  ceases  and  they  become  agglutinated  into  clumps 


FIG.  97. — Application  of  the  serum-reaction  to  typhoid  bacilli.  A  shows 
the  distribution  of  the  bacilli  before  the  reaction.  It  is  to  be  remembered  that 
they  are  motile  and  their  positions  may  change  continually.  B  shows  clump- 
ing of  the  motionless  bacilli  after  mixture  with  the  serum  of  a  case  of  typhoid 
fever.  (Diagrammatic.} 


or  masses  (see  Agglutinins,  Bacterial  Poisons,  page  190).  The  bacilli  may 
eventually  undergo  disintegration  into  granular  material  (see  Lysins,  Bacterial 
Poisons,  page  193).  This  reaction  rarely  takes  place  with  the  blood-serum 
of  healthy  persons  or  of  those  suffering  with  other  diseases,  nor  when  the  blood- 
serum  of  a  typhoid  fever  case  is  mixed  with  motile  bacteria  other  than  typhoid 
bacilli:  It  has  been  observed  in  the  blood-serum  of  an  infant  born  while  the 
mother  was  convalescing  from  typhoid  fever. 

The  agglutinating  substance  has  been  found  in  blister-serum  and  in  the 
milk  of  typhoid  cases,  in  fluids  from  the  serous  cavities  and  inflammatory  and 
edematous  areas  in  variable  amounts,  and  occasionally  in  urine,  bile  and  tears. 

The  reaction  may  be  obtained  by  adding  blood-serum  to  a  young  bouillon- 
culture  of  typhoid  bacilli  kept  in  the  incubator,  when  the  occurrence  of  agglutina- 

*This  test  is  often  known  as  the  "  Widal  reaction."  For  a  history  and  general 
discussion  of  the  subject  see  Durham.  Journal  of  Experimental  Medicine. 
Vol.  V.  p.  353. 


PATHOGENIC    BACTERIA.  377 

tion  becomes  manifest  by  the  collection  of  the  bacteria  into  visible  masses  or 
flocculi,  which  form  a  sediment.  Most  investigators  prefer  to  watch  the  results 
under  the  microscope,  using  an  ordinary  slide,  or,  better,  the  hanging-drop. 
Young  cultures — less  than  twenty-four  hours  old — in  bouillon,  and  kept  in  the 
incubator,  may  be  used,  or,  better,  cultures  kept  at  room-temperature  for 
twenty-four  hours.  Johnston  and  McTaggart  recommend  that  the  bouillon 
cultures  be  freshly  made  each  time  from  stock  cultures  on  agar,  which  need 
only  occasionally  be  transplanted.  Certain  stocks  of  typhoid  bacilli  seem  especi- 
ally suited  to  this  reaction,  and  such  a  stock  should  be  secured. 

Blood-serum,  blister-serum,  fresh  blood  and  dried  blood  have  all  been  used 
with  success.  Blood  dried  on  unglazed  paper  or  cover-glasses  as  proposed 
by  Wyatt  Johnston  is  extremely  convenient.  To  perform  the  test  the  drop 
of  dried  blood  is  mixed  on  the  paper  with  sterilized  bouillon  or  normal  salt 
solution,  and  portions  of  the  suspension  of  the  blood-serum  obtained  in  this  way 
are  tested  upon  the  typhoid  cultures.  The  objection  to  this  procedure  lies  in 
the  dificulty  of  securing  an  accurate  dilution.  An  approximate  knowledge  of  the 
degree  of  dilution  may  be  acquired  by  mixing  drops  of  dried  blood  of  known 
volume  with  definite  amounts  of  water,  and  observing  the  tints.  These  should 
be  kept  in  mind  as  standards.  The  dilution  may  be  measured  with  the  hemoglo- 
binometer  or  with  the  pipette  of  the  hemocytometer.  The  New  York  Board  of 
Health  have  found  blister-serum  satisfactory  and  easy  to  obtain.  A  little  of  the 
diluted  serum  is  mixed  on  the  cover-glass  with  a  definite  amount  of  the  fresh 
bouillon-culture,  and  is  examined  as  a  hanging-drop.  In  a  short  time  the 
characteristic  clumping  and  loss  of  motility  occur.  At  the  same  time  a  drop 
of  the  culture  alone,  and  a  drop  of  the  culture  mixed  with  normal  serum,  similarly 
diluted,  should  be  examined  as  controls.  The  dilutions  finally  resulting  after 
mixing  with  the  drop  of  culture,  when  placed  under  the  microscope,  vary 
from  one  part  of  serum  in  30  to  i  in  50.  The  higher  dilution  at  which  clumping 
takes  place  the  more  definite  the  result.  The  time  within  which  the  reaction 
occurs  varies  from  a  few  minutes  to  about  one.  With  little  dilution  the  time 
should  be  short;  with  greater  dilution  it  may  be  longer.  Both  cessation  of 
motility  and  clumping  should  take  place.  In  a  positive  case  the  cessation  of 
motion  and  the  clumping  of  the  bacilli  should  be  complete.  Normal  blood 
sometimes  exhibits  agglutinative  properties  in  some  degree.  Some  cultures 
show  a  tendency  to  clump  without  the  addition  of  any  blood  serum,  but 
such  sources  of  error  are  checked  by  the  control  tests  described.  If  the 
reaction  in  any  case  is  not  satisfactory,  it  should  be  tried  with  a  higher  dilu- 
tion, i  to  50  or  more,  and  the  result  should  be  positive  if  the  case  is  a  genuine 
case  of  typhoid  fever. 

The  agglutinating  power  usually  appears  in  the  blood  between  the  seventh 
day  and  the  end  of  the  third  week  of  the  disease;  it  may  be  seen  earlier;  it  is 
often  delayed  and  appears  late.  The  test  frequently  has  to  be  repeated  when  the 
first  result  is  doubtful  or  negative.  Reports  indicate  that  the  method  is  a  great 
aid  in  the  diagnosis  of  typhoid  fever,  though  not  infallible. 

The  use  of  dead  cultures  for  the  agglutination  reaction  has  been  recommended 
by  various  authors,  and  Ficker  has  placed  his  so-called  "Diagnosticum,"  a 
suspension  of  dead  typhoid  bacilli,  on  the  market.  For  this  method  of  diagnosis 
Ruediger*  makes  the  following  recommendation:  Inoculate  a  large  flask — 
100  or  1,000  c.c. — with  the  typhoid  bacillus  and  incubate  at  36°  C.  for  24  hours. 
At  the  end  of  this  time  add  i  c.c.  of  formalin  for  every  100  c.c.  of  broth.  This 
is  now  ready  for  use,  and  can  be  preserved  for  many  months.  It  must  be  shaken 
up  every  time  before  use  to  distribute  the  organisms.  Ruediger  further  advises 
that  in  making  the  test  four  drops  of  blood  be  mixed  with  2  c.c.  of  a  2  per  cent,  for- 

*Journ.  Inject.  Dis.  Vol.  I.   1904.     pp.  236-240. 


378  MANUAL    OF    BACTERIOLOGY. 

malin  solution,  and  that  i  c.c.  of  this  diluted  blood  be  added  to  4  c.c.  of  the 
dead  culture.  This  mixture  is  set  aside  along  with  a  control  tube  made  with 
normal  ,blood  or  with  distilled  water.  If  agglutination  takes  place,  the  dead 
organisms  settle  down  in  a  flocculent  precipitate  in  an  hour  or  two. 

By  the  use  of  a  medium  consisting  of  90  c  c.  of  ox-bile,  10  c.c.  of  glycerin,  and 
2  grams  of  peptone.  Coleman  and  Buxton*  have  come  to  the  conclusion  that  the 
typhoid  bacillus  is  present  in  the  blood  in  every  case  of  typhoid  fever  throughout 
its  course.  These  investigators  distribute  this  medium  into  small  flasks,  20  c.c. 
into  each,  which  are  then  sterilized.  The  blood  to  be  examined  is  drawn  by 
means  of  an  all  glass  syringe-from  the  vein  at  the  bend  of  the  elbow,  and  3  c.c. 
of  it  introduced  into  each  of  three  of  the  flasks  of  ox-bile  medium.  After 
incubation  over  night,  streak  cultures  are  made  from  the  surface  of  the  liquid 
in  the  flasks  on  the  surface  of  litmus-lac tose-agar  plates.  Further  procedures 
for  the  identification  of  the  organism  are  those  already  given. 

Considerable  experience  is  necessary  to  acquire  the  judgment  needed  in 
using  this  test. 

The  agglutinating  power  becomes  lessened  after  recovery,  and  usually  is 
wanting  at  the  end  of  a  year.  Rarely  it  may  be  present  for  a  longer  time,  a 
fact  that  is  to  be  borne  in  mind  in  making  a  diagnosis. 

Various  observers  have  obtained  the  Widal  reaction  with  serum  patients 
suffering  from  other  diseases  than  typhoid. 

Typhoid  bacilli  have  frequently  been  obtained  from  the 
stools  of  cases  of  the  disease,  but  they  are  isolated  only  with 
considerable  difficulty.  At  autopsies  they  are  best  cultivated 
from  the  spleen,  in  which,  however,  it  is  to  be  remembered, 
the  Bacillus  coli  communis  may  also  be  present.  Cultures 
made  from  the  blood,  where  several  cubic  centimeters  are  taken, 
show  that  a  few  bacilli  occur  in  the  blood  in  a  large  proportion 
of  cases  of  the  disease — probably  in  a  majority.  Typhoid 
bacilli  appear  in  the  urine  in  about  20  per  cent,  of  all  cases, 
and  the  examination  of  urine  for  them  has  been  used  in 
diagnosis.  The  bacilli  often  occur- in  the  gall-bladder.  They, 
as  well  as  the  colon  bacillus,  have  been  found  inside  of  gall- 
stones, and  have  been  supposed  to  be  one  of  the  causes  for 
the  formation  of  gall-stones. f  They  may  remain  present  in 
the  gall-bladder  or  in  the  urinej  long  after  convalescence  from 
the  disease.  They  have  been  demonstrated  in  the  "rose 
spots"  on  the  abdomen.  They  may  be  present  in  the  lesions 

*Reprint  Am.  Jour.  Med.  Sci.    June,  1907. 

fPratt.  American  Journal  Medical  Sciences.  Vol.  CXXII.  1901.  Also 
Kramer.  Journ.  Exper.  Med.  Vol.  IX.  1907.  pp.  319-323. 

JM.  W.  Richardson.     Journal  Experimental  Medicine.     Vol.  IV.     1899. 


PATHOGENIC    BACTERIA.  379 

of  the  pneumonia,*  which  frequently  complicates  typhoid 
fever,  and  may  appear  in  the  sputum.  In  times  of  epidemic 
of  the  disease  they  may  be  present  in  the  stools  of  persons  who 
show  no  symptoms  of  typhoid  fever.  Nieterf  found  13 
such  typhoid  fever  bacillus  carriers  in  a  certain  German 
insane  asylum. 

Inoculation  experiments  in  animals  have  not  been  very 
satisfactory.  With  a  few  possible  exceptions,  anatomical 
lesions  resembling  those  of  typhoid  fever  have  not  been  pro- 
duced by  the  inoculation  of  typhoid  bacilli  into  animals.  The 
injection  of  cultures  into  animals  may  produce  death,  but  it  can 
usually  be  shown  to  have  resulted  from  the  poisons  contained 
in  the  cultures. 

BesredkaJ  obtained  endotoxins  from  the  bacilli  of  typhoid  fever,  plague  and 
dysentery  by  triturating  with  salt  to  an  impalpable  powder,  adding  water,  drop 
by  drop,  allowing  to  remain  over  night,  and  with  the  typhoid  bacilli  heating 
at  60  to  62°  C.  for  two  hours,  allowing  to  settle  and  decanting  the  liquid  which 
contains  the  endotoxin  with  the  plague  and  dysentery  bacilli  the  separation  is 
affected  without  heat  by  centrifugalizing. 

\ 

Typhoid  fever  is  rare  during  the  first  two  years  of  life.  It 
frequently  attacks  young  and  robust  men.  The  causes  that 
bring  about  susceptibility  to  infection  are  not  known. 

The  principal  lesion  in  typhoid  fever  lies  in  the  Peyer's 
patches  of  the  lower  part  of  the  small  intestines;  the  mes- 
enteric  lymph-nodes  and  spleen  also  are  swollen.  The  typhoid 
bacillus  may  be  demonstrated  in  sections  of  the  walls  of  the 
diseased  portion  of  the  intestines.  Cases  are  recorded  in 
which  no  lesions  were  found  in  the  intestines,  but  where  the 
typhoid  bacilli  were  widely  spread  through  the  organs  of  the 
body,  and  which  therefore  represented  typhoid  septicemia. 

Periostitis  and  osteomyelitis,  which  are  not  uncommon 
sequelae  of  typhoid  fever,  may  be  caused  by  typhoid  bacilli. 

*Robinson.     Journ.  Inject.  Dis.  Vol.  II.     1905.     p.  49&- 
fMunchner.     Med.  Wochenschr.     Aug.  13,  1907.     LIV.  No.  33. 
%Bull  de  I'Inst.  Past.     Vol.  IV.,  No.  13.     July  15,  1906.  pp.  586-587. 


380  MANUAL    OF    BACTERIOLOGY. 

Ordinary  suppuration  may  be  produced  by  the  typhoid  bacillus 
but  most  suppurative  affections  during  or  following  typhoid 
fever  are  mixed  infections,  or  are  due  to  the  ordinary  pyogenic 
bacteria. 

That  typhoid  fever  is  transmitted  chiefly  through  the 
medium  of  water,  has  long  been  held,  though  there  are  some 
who  now  regard  other  modes  of  transmission  of  equal  or 
more  importance. 

In  Washington,  D.  C.,  there  was  a  marked  increase  in  the 
number  of  cases  of  typhoid  fever  after  the  introduction  of 
the  water  nitration  plant.*  After  the  introduction  of  the  filter 
plant  the  purity  of  the  water  as  shown  by  the  number  of 
bacteria  was  in  inverse  proportion  to  the  death-rate  from 
typhoid  fever.f  RosenauJ  attributes  the  typhoid  fever  in  the 
District  to  three  causes:  To  importation,  to  infected  milk,  and 
to  contact.  He  attributed  n  per  cent,  of  cases  during  1906 
and  9  per  cent,  during  the  summer  of  1907  to  milk;  7  per  cent, 
during  1906,  19  per  cent,  during  the  summer  of  1907  to  con- 
tact; 15  per  cent,  during  1906,  25  per  cent,  during  the  summer 
of  1907  to  importation.  It  is  sometimes  conveyed  by  milk,  by 
green  vegetables  and  by  oysters.  Infection  through  the  medium 
of  dust  and  by  the  hands  and  clothing  probably  occurs,  but 
not  commonly.  Under  certain  circumstances  the  bacilli 
may  be  carried  by  flies,§  and  it  is  consequently  of  the 
greatest  importance  in  preventing  the  spread  of  the  disease 
to  prevent  flies  from  having  access  to  the  excreta  of  typhoid 
fever  patients.  In  caring  for  cases  of  typhoid  fever  the  stools, 
urine,  sputum  and  linen  should  be  disinfected.  Persons 
handling  the  patient  should  wash  and  disinfect  their  hands 
after  every  contact  with  the  patient. 

*Ed.  Journ.  Am.  Med.  Assn.     Vol.  XL VIII.      No  17.     April  27..  1907.  p.  135. 
fCosby.     Wash.  Med.  Ann.     Report  of  meetings  of  the   Medical  Society, 
Washington,  D.  C.,  Feb.  19,  and  26,  1908.     p.  135. 
%  Ibid. 
§Vaughan.     Philadelphia  Medical  Journal.     June  9,  1900. 


PATHOGENIC    BACTERIA.  381 

W  hile  the  idea  formerly  entertained  that  sewer  gas  is  itself 
the  cause  of  typhoid  fever,  Horrocks*  points  out  that  the 
bubbles  of  gas  in  sewage  may  be  the  means  of  spreading  the 
contagium. 

The  injection  of  typhoid  bacilli  which  have  been  killed  by  heat  has  been 
resorted  to  as  a  preventive  measure  in  a  large  number  of  cases  in  the  British 
army.  The  results  appear  to  have  been  partially  successful,  but  the  method 
is  still  in  an  experimental  stage. 

Richardsonf  sums  up  his  results  with  the  use  of  specific  sera,  filtrates  from 
cultures,  and  non-toxic  extracts  from  cultures  (Vaughan)  approximately  as 
follows:  Seventy-four  cases  were  treated  by  various  methods  with  serum 
prepare  by  inoculating  horses  with  typhoid  cultures  as  in  the  method  of  pro- 
ducing diphtheria  antitoxin;  35  cases  were  treated  with  the  filtrates  through 
porcelain  filters  of  cultures  of  typhoid  bacilli;  21  cases  were  treated  with  non- 
toxic  extracts  of  the  typhoid  bacillus  prepared  by  Vaughan.  All  these  methods 
increase  the  tendency  to  relapse  unless  the  Vaughan  non-toxic  treatment  is 
kept  up  into  the  stage  of  convalescence.  The  use  of  serum  has  no  advantages 
over  the  filtrates  and  extracts  upon  the  course  of  the  disease.  Filtrates  may 
produce  chills,  rise  of  temperature  and  pulse,  followed  by  a  general  improve- 
ment in  the  clinical  aspect.  The  non-toxic  residue  of  Vaughan  seems  to  make  the 
typhoid  process  longer,  but  milder,  and  to  prevent  relapse  in  convalescence. 

Bacillus  coli  communis  (often  called  simply  the  colon 
bacillus,  Bacterium  coli  commune  of  Escherich,  and  Bacillus 
pyogenes  fcetidus  of  Passet,  who  obtained  it  from  foul  pus; 
probably  the  same  as  Bacillus  Neapolitanus  of  Emmerich).— 
A  bacillus  with  rounded  ends,,  frequently  of  a  short,  oval  form, 
when  it  may  be  difficult  to  distinguish  from  micrococci;  often 
longer,  even  forming  threads.  It  is  slightly  motile,  having 
several  flagella.  It  does  not  form  spores.  It  stains  with 
the  ordinary  aniline  dyes,  but  not  by  Gram's  method.  It  is  a 
facultative  anaerobe.  It  grows  well  at  the  room-temperature, 
but  more  rapidly  in  the  incubator.  It  does  not  liquefy  gelatin. 
In  gelatin  plates  the  surface  colonies  are  of  a  bluish-white  color; 
the  centers  are  denser  than  the  borders,  which  are  translucent. 
It  usually  grows  more  rapidly  in  gelatin  than  the  bacillus  of 
typhoid  fever.  Its  growths  in  other  media  are  mostly  whitish. 
Bouillon  becomes  clouded.  Nitrates  are  reduced  to  nitrites. 

*Ed.  in  Journ.  Am.  Med.  Assn.    Vol.  XLVIIL,  June  i,  1907.     p.  1869. 
t Annual  Meeting  of  the  Massachusetts  Medical  Society.     Boston.     June  n, 
TOO;. 


382  MANUAL    OF    BACTERIOLOGY. 

In  peptone  solution  it  forms  indol.  On  potato  it  forms  an 
abundant  visible  growth  from  cream  color  to  pale  brown. 
Milk  becomes  acid  and  is  usually,  but  not  always,  coagu- 
lated slowly.  It  causes  the  development  of  gas  and  acid  in 
media  containing  dextrose  or  lactose.  In  media  containing 
neutral  red  the  colon  bacillus  produces  a  yellow  color  with  a 
green  fluorescence.  Differential  points  between  the  bacillus  of 
typhoid  fever  and  the  Bacillus  coli  communis  are  as  follows: 


•v  ->      .  «•• 

.-.I- 


^te-s  *• 

*?     »'f    f 

v>»*?*»  ^  .«*••  ^ 

y*  «%<     »  %»/.  ->  ,'%  .  * 

>'i-  -x  i*     *-t     ^    -ir  ^ 

#«3A--      v  ^      ^  rx 


i7Cf 


FIG.  98. — Bacillus  coli  communis.     (X  1000.) 

ist.  The  typhoid  bacillus  is  actively  motile;  the  colon 
bacillus  less  actively  motile. 

2d.  The  typhoid  bacillus  has  numerous  flagella  which  rise 
from  all  parts  of  the  surface;  the  colon  bacillus  has  a  smaller 
number  of  flagella. 

3d.  The  colonies  of  the  typhoid  bacillus  in  gelatin  develop 
more  slowly  than  those  of  the  colon  bacillus. 

4th.  The  superficial  colonies  of  the  typhoid  bacillus  on  gela- 
tin, plates  are  less  dense  than  those  of  the  colon  bacillus. 

5th.  In  media  containing  dextrose  or  lactose  the  typhoid 


PATHOGENIC    BACTERIA.  383 

bacillus  does  not  produce  fermentation  with  gas  and  the  colon 
bacillus  does  produce  gas  in  such  media. 

6th.  The  typhoid  bacillus  produces  a  very  slight  acid  reac- 
tion without  coagulation  in  milk,  and  the  colon  bacillus  pro- 
duces a  strong  acid  reaction  with  coagulation. 

yth.  In  peptone  solution  the  typhoid  bacillus,  as  a  rule, 
produces  no  indol,  and  the  colon  bacillus  produces  indol. 


-.  ,  *• 


FIG.  99. — Bacillus  coli  communis  with  flagella,  stained  by  Van 
Ermengem's  method.     (X  1000.) 

8th.  The  typhoid  bacillus  usually  produces  an  invisible 
growth  on  potato,  the  colon  bacillus  a  visible  growth. 

gth.  The  typhoid  bacillus  is  said  not  to  reduce  neutral 
red  in  media,  and  the  colon  bacillus  to  change  it  to  a  yellow 
color. 

To  these  may  be  added  the  growth  of  the  two  organisms 
on  special  media  like  those  of  Wttrtz,  of  Eisner,  of  Hiss  and 
of  Drigalsky  and  Conradi  and  the  application  of  the  serum- 
reaction. 

Injections  of  cultures  of  the  Bacillus  coli  communis  into 


384  MANUAL    OF    BACTERIOLOGY. 

animals  produce  variable  and  uncertain  results.  Subcutane- 
ous injection  may  lead  to  pus-formation;  in  rabbits  and 
guinea-pigs  injections  may  produce  death  apparently  from 
poisons  introduced.  With  the  blood  of  immunized  animals  a 
serum-reaction,  similar  to  that  described  for  typhoid  fever, 
may  be  demonstrated. 

Concerning-  the  occurrence  of  the  Bacillus  coli  communis 
in  the  intestine  of  man  see  page  164.* 

At  autopsies  on  human  subjects  the  great  viscera  are  often 
found  to  have  been  infected  by  the  colon  bacillus,  usually  when 
some  lesion  of  the  intestine  existed  simultaneously,  but  in  most 
cases  without  having  produced  much  apparent  damage  to  the 
organs  invaded.  The  Bacillus  coli  communis  frequently 
occurs  in  mixed  infections,  as  in  wounds,  inflammations  and 
abscesses.  It  is  often  found  in  the  peritoneum  in  peritonitis,  in 
the  pus  in  appendicitis,  and  in  the  urine  in  cystitis;  it  fre- 
quently occurs  in  the  interior  of  gall-stones  with  whose  for- 
mation it  may  be  connected,!  as  first  pointed  out  by  Welch. 

There  is  a  large  number  of  more  or  less  closely  related 
organisms  which  go  by  the  name  of  the  "colon  group."  The 
limits  of  the  colon  group  are  extremely  ill-defined.  J 


Detection  of  Bacillus  Coli  Communis  in  Water.  —  To  each  of  a  number  of 
fermentation-tubes  containing  i  per  cent,  dextrose-bouillon  add  some  of  the 
suspected  water  (o.i  to  i  c.c.  or  more).  Place  in  the  incubator.  Each  day 
mark  the  amount  of  gas  that  has  formed  in  the  closed  arm.  After  two  days 
B.  coli  communis  should  render  the  bouillon  strongly  acid  and  produce  about 
50  per  cent,  of  gas  (30  to  70  per  cent,  according  to  different  writers).  The 
gas  is  approximately  H  two  parts,  and  CO2  one  part  (see  page  132).  From 
tubes  showing  these  characters  plates  may  be  made  and  the  usual  tests  for 
the  colon  bacillus  applied.!  (See  Part  IV.)  Stokes  recommends  adding  the 
water  to  fermentation  tubes  containing  i  per  cent,  lactose  -bouillon  and  neutral 
red  (10  c.c.  of  a  5  per  cent,  solution  of  neutral  red  to  a  liter  of  bouillon);  if 
the  colon  bacillus  is  present,  30  per  cent,  to  50  per  cent,  of  gas  is  formed  (con- 


*See  also  Moore  and  Wright.     Bacillus  coli  communis  in  the  Domesticated 
Animals.     American  Medicine.     March  29,  1902. 

fLartigau.     Journal  American  Medical  Association.     April  12,  1902. 


jRivas.     Journ.  Med.  Res.     XV.     1906.     pp.  397-509.' 
§Theobald  Smith.     American  Journal  Medical  Sciences. 


Vol.  CX.     1895. 


PATHOGENIC    BACTERIA.  385 

sisting  of  one  part  of  carbon  dioxide  and  two  parts  of  hydrogen),  and  the  neu- 
tral red  in  the  closed  arm  changes  to  a  yellow  color.* 

Jacksonf  advises  the  use  of  bile  as  an  inhibiting  agent  for  the  bacteria  other 
than  the  intestinal  bacteria  found  in  water.  The  restraining  action  of  the  bile 
is  due  to  the  colic  acid  radical  of  the  bile.  Jackson  recommends  that  undiluted 
ox-bile  be  employed.  This  is  sterilized  as  soon  as  it  is  drawn,  and  may  then 
be  kept  in  stock.  When  used  it  should  be  decanted  or  filtered,  and  one  per  cent, 
of  lactose  previously  dissolved  in  a  small  amount  of  water  should  be  added. 
It  should  then  be  distributed  into  fermentation  tubes  140  mm.  long  by  15  mm. 
in  diameter  having  an  elongated  bulb  38  mm.  in  the  shortest  diameter.  Instead 
of  fresh  bile,  no  grams  of  dried  bile  and  10  grams  of  lactose  dissolved  in  i  liter 
of  distilled  water  may  be  employed. 

SwinJ  recommends  this  medium  particularly  for  sewage  and  for  badly 
contaminated  water. 

The  following  scheme  for  the  detection  of  B.  coli  in  water  has  been  recom- 
mended by  Longley  and  Warren.  § 

Preliminary  incubation  in  dextrose-broth  fermentation  tubes  at  40°  C.  for  24 
hours.  Those  showing  gas  are  to  be  plated  on  litmus-lactose-agar.  No  note 
need  be  made  of  the  reaction  nor  of  the  amount  of  gas.  The  above  plates  are 
to  be  incubated  at  40°  C.  for  18  or  24  hours,  and  inoculations  made  on  agar 
slants  from  any  red  colonies  that  may  be  found.  The  agar  slants  shall  be 
incubated  at  40°  C.  for  24  hours,  and  further  tests  made  in  dextrose-broth  fer- 
mentation tubes.  Tubes  developing  no  gas  are  noted  as  negative.  Milk 
tubes  should  also  be  inoculated  from  the  agar  slants  and  incubated  at  40°  C. 
for  two  days,  and  examined  for  coagulation  both  days.  Failure  to  coagulate 
or  digestion  of  casein  after  coagulation  are  to  be  regarded  as  negative.  Culti- 
vation for  two  days  at  40°  C.  in  nitrate-broth  and  testing  for  the  presence  of 
nitrites  at  end  of  this  time.  The  presence  of  nitrites  is  regarded  as  positive. 
Cultures  from  the  slant  agar  in  peptone  broth  are  incubated  for  3  days  at 
40°  C.  and  then  tested  for  the  presence  of  indol.  The  presence  of  indol  is  regarded 
as  positive.  The  above  statement  is  a  somewhat  condensed  statement  of 
Longley  and  Warren's  scheme. 

Paracolon  or  paratyphoid  bacilli  are  the  names  applied  to  certain  members 
of  the  colon  group  which  have  recently  been  shown  to  be  pathogenic  to  man. 
They  may  produce  clinical  symptoms  resembling  typhoid  fever  of  a  mild  and 
atypical  form,  but  Wells  and  Scott  ||  found  that  there  is  little  to  distinguish  the 
pathological  lesions  produced  by  the  paracolon  bacillus  from  other  septicemias. 
The  intestinal  lesions  are  quite  variable,  but  there  are  no  changes  in  the  Peyer's 
plaques  or  solitary  follicles.  The  intestinal  glands  are  little  or  not  at  all  affected. 
The  ulcers  of  the  intestines  are  not  always  present  and  when  they  are  they 
differ  from  those  found  in  typhoid  fever.  Probably  they  may  occur  with  typhoid 
fever  in  mixed  and  secondary  infections.  Characteristic  lesions  have  not  yet 
been  observed.  The  most  constant  change  is  splenic  enlargement.  The 
affection  is  rarely  fatal.  The  bacilli  have  been  found  in  the  blood,  spleen, 
liver,  gall-bladder  and  urine.  Like  typhoid  and  colon  bacilli  they  are  motile, 
have  flagella,  are  not  stained  by  Gram's  method  and  do  not  liquefy  gelatin. 
They  ferment  dextrose  and  maltose,  producing  acid  and  gas.  They  do  not 
ferment  lactose.  Milk  at  first  becomes  acid,  later  it  becomes  alkaline,  and 
is  not  coagulated.  On  potato  a  slight  visible  growth  occurs.  Media  contain - 

^Journal  of  Infectious  Diseases.     I.     341. 

^Journal  Infect.  Diseases.     Supplement  No.  3.     May,  1907.     pp.  30-32. 

llbid.     pp.  32-38. 

\Journ.     Inject.    Dis.    Vol.  IV.   1907,  pp.  397-415. 

\\Journ.  Infect.  Dis.  Vol.  I.  1904.     pp.  72-90. 

25 


386  MANUAL    OF    BACTERIOLOGY. 

ing  neutral  red  become  yellow,  as  with  B.  coli  communis,  but  more  slowly,  and 
the  red  color  sometimes  returns.  In  respect  to  the  fermentation  of  saccharose 
and  the  formation  of  indol  reports  differ;  both  are  usually  negative.  The 
blood  of  the  patient  agglutinates  the  bacilli.  But,  as  among  the  closely  related 
members  of  this  group  mutual  reactions  are  sometimes  seen,  this  test  is  not 
to  be  considered  invariable.*  Several  bacilli  allied  to  the  above  are  known. 
The  Bacill  usenteritidis  of  Gaertner  is  a  related  form  which  has  been  found ; 
in  cases  of  meat-poisoning. 

Bacillus  Lactis  Aerogenes  (Bacillus  aerogenes). — A  ba- 
cillus having  a  form  similar  to  that  of  the  colon  bacillus,  de- 
scribed as  being  larger  and  plumper.  In  the  main  its  proper- 
ties are  similar  to  those  of  the  colon  bacillus.  Its  colonies 
are  more  circumscribed  and  elevated  than  those  of  the  colon 
bacillus.  It  is  non-motile.  It  coagulates  milk  more  rapidly 
than  the  colon  bacillus.  It  produces  gas  upon  potato  more 
rapidly  than  the  colon  bacillus,  and  more  abundantly.  It  was 
first  described  by  Escherich,  who  discovered  the  colon  bacillus, 
assigning  the  Bacillus  lactis  aerogenes  rather  to  the  upper  part 
of  the  small  intestine,  and  the  colon  bacillus  to  the  lower 
portion.  According  to  Kruse,  the  Bacillus  lactis  aerogenes 
and  its  relatives  differ  from  the  Bacillus  coli  communis  chiefly 
in  lacking  motility.  Like  the  colon  bacillus  it  has  been  found 
many  times  in  the  urine  in  cystitis.  See  also  B.  acidi  lactici, 
page  273. 

Bacillus  Dysenteriae  (Shiga).— A  bacillus  with  rounded 
ends,  of  the  size  and  shape  of  typhoid  and  colon  bacilli,  seldom 
forming  threads.  Most  observers  have  found  it  non-motile. 
Vedder  and  Duval  have  demonstrated,  flagella.  The  bacillus 
does  not  form  spores.  It  may  be  stained  with  the  ordinary 
aniline  dyes;  it  does  not  stain  by  Gram's  method.  It  is  a 
facultative  anaerobe.  It  grows  at  ordinary  temperatures,  but 
better  in  the  incubator.  It  grows  on  the  usual  culture-media, 
but  more  slowly  than  B.  coli  communis.  The  growths  are 

*Crushing.     Bulletin  Johns  Hopkins  Hospital.     July-August,  1900.     Strong,  j 
Ibid.     May,    1902.     Johnstone,   Hewlett   and   Longcope.     American  Journal 
Medical   Sciences.     August,    1902.     Libman    and   Buxton.     Journal  Medical  , 
Research.     Vol.  VIII.     1902. 


PATHOGENIC    BACTERIA.  387 

whitish.  Colonies  on  gelatin  plates  resemble  those  of  the 
typhoid  bacillus.  Bouillon  is  diffusely  clouded;  a  precipitate 
may  form,  but  no  pellicle.  Indol  is  not  produced.  Milk 
becomes  acid  and  is  not  coagulated.  On  potato  a  thin  pale 
layer  forms  which  may  become  light  brown.  No  gas  is  formed 
in  media  containing  glucose  or  lactose. 

Neutral-red  agar  is  not  changed.  From  the  feces  the 
bacillus  is  best  cultivated  on  agar  plates,  in  the  incubator. 
Colonies  of  B.  coli  communis  are  often  more  numerous  than 
those  of  the  dysentery  bacillus.  The  colonies  which  develop 
in  twenty-four  hours  are  likely  to  be  colonies  of  B.  coli  com- 
munis. The  position  of  these  may  be  marked  on  the  glass 
with  a  pencil.  Those  which  appear  later  are  to  be  planted  in 
dextrose-agar.  If  gas  develops,  they  are  not  the  bacilli  of 
dysentery;  otherwise  they  are  to  be  studied  and  identified 
by  the  cultural  and  other  tests  mentioned  above,  and  by  the 
agglutination  reaction. 

They  have  been  found  in  the  intestine  and  the  discharges 
of  acute. and  epidemic  dysentery  in  various  climates  and  coun- 
tries, including  the  United  States.  Thus  far  their  dissemina- 
tion in  the  blood  and  distant  organs  has  not  been  demon- 
strated. The  lesion  of  this  form  of  dysentery  consists  of  a 
severe  acute  inflammation  of  the  colon,  frequently  with  necrosis 
of  the  surface  and  the  formation  of  pseudomenbrane.  Ulcer- 
ation  may  occur,  but  is  usually  superficial.  Duval  and 
Bassett  found  the  bacillus  of  dysentery  in  the  stools  of  infants 
having  summer  diarrhea;  but  Collins*  failed  to  find  B.  dysen- 
teriae  or  paradysenteriae  in  infants  suffering  from  acute  and 
subacute  diarrhea. 

The  introduction  of  pure  cultures  into  animals  by  way  of 
the  alimentary  canal  has  sometimes  been  followed  by  a  cer- 
tain amount  of  diarrhea,  but  it  does  not  appear  that  dysen- 
tery, as  it  occurs  in  man,  has  been  reproduced.  Most  labor- 

*  Jo-urn.  Inject.  Dls.  Vol.  II.     1905.     pp.  620-626. 


388 


MANUAL    OF    BACTERIOLOGY. 


atory  animals  are,  however,  very  sensitive  to  the  injection  into 
the  tissues  or  veins  of  cultures,  living  or  dead.  They  show  the 
lesions  produced  by  various  toxins,  but  nothing  of  a  character- 
istic nature'. 

The  bacillus  is  agglutinated  by  the  patient's  blood,  but 
often  only  late  in  the  disease  and  apparently  not  in  all  cases. 
This  test  seems  to  have  only  a  limited  value  in  clinical  diag- 


FIG.  100. — Spirillum  of  cholera.*     (X  1000.) 

nosis.  Many  prefer  to  secure  the  reaction  in  a  test-tube. 
The  dilutions  used  vary  greatly  (from  i  in  20  to  i  in  100). 
Immunized  animals  develop  the  agglutinins  in  the  blood. 
Results  of  experiment  made  for  the  production  of  a  curative 
serum  are  encouraging. 

Torreyt  comes  to  the  conclusion  that  the  group  of  dysentery 
organisms  is  a  large  and  varied  one  which  may  be  divided 
into  two  groups:  the  Shiga-Kruse  group  on  the  one  hand  and 

*The  specimen  from  which  this  photograph  was  made  was  stained  for 
flagella.  A  field  was  selected  where  the  organisms  failed  to  show  flagella.  But 
the  method  of  stai.iing  probably  accounts  for  the  bacteria  appearing  somewhat 
thicker  than  they  usually  do  in  preparations. 

\Journ.  Exper.  Med.     Vol.  VII.     Feb.-Nov.,  1905.     pp.  365-384. 


PATHOGENIC    BACTERIA.  389 

the  manite-fermentcrs  on  the  other.  Among  the  manite- 
fermenters,  of  which  bacillus  "Y"  of  Hiss  and  Russell  and 
the  Flexner-Manila  bacillus  are  the  type,  there  is  great  varia- 
tion in  cultural  properties,  but  have  this  in  common  that  they 
all  produce  the  same  change  in  litmus-milk.  Flexner  suggests 
that  these  organisms  may  be  occasionally  or  constantly 
present  in  the  normal  intestines.  Duval  found  them  in  the 
intestinal  contents  of  children  suffering  from  mild  summer 
diarrhea.  On  the  other  hand  the  Shiga-Kruse  type  from 
some  twenty  different  sources  all  reacted  alike  in  the  various 
culture-media  and  all  agglutinated  alike  with  various  sera. 
According  to  W.  H.  Park,  some  of  the  manite-fermenters  form 
indol  which  the  bacillus  of  Shiga  does  not;  they  also  differ 
from  it  in  their  agglutination  reactions.* 

While  it  seems  probable  that  the  Shiga  bacillus,  and  its 
congeners  cause  certain  forms  of  dysentery,  Jiirgeus  states  that 
there  is  evidence  to  show  that  there  are  epidemics  of  dysentery 
which  are  caused  by  organisms  which  can  scarcely  be  identified 
with  them.t 

It  must  also  be  borne  in  mind  that  epidemics  of  dysentery 
occur  mainly,  though  not  exclusively,  in  tropical  countries 
which  are  caused  by  amebae  (see  page  430). 

Bacillus  Pseudodysentericus.— MullerJ  gave  the  name  B.  pseudodysenter- 
icus  to  a  type  of  organisms  bearing  all  the  cultural  characteristics  and  patho- 
genic properties  for  experiment  animals  which  are  shown  by  B.  dysenteriae,  and 
differing  from  this  organism  or  group  of  organisms  in  one  respect  only;  that  is  in 
the  failure  to  agglutinate  with  blood-serum  of  persons  suffering  with  dysentery. 
While  stating  that  this  classification  of  the  organism  is  purely  arbitrary  and 

*Shiga.  Centralblatt  }ur  Bakteriologie.  Bd.  XXIV.  1898.  Flexner. 
Philadelphia  Medical  Journal.  September  i,  1900.  Vedder  and  Duval. 
Journal  Experimental  Medicine.  Vol.  VI.  Gay.  University  of  Pennsylvania 
Medical  Bulletin.  November,  1902.  Duval  and  Bassett.  American  Medicine. 
Vol.  IV.  p.  417.  1902.  Park  and  Carey.  Journal  Medical  Research 
Vol.  IX.  1903.  Strong  and  Musgrove.  Journal  American  Medical  Associa- 
tion. Vol.  XXXV.,  p.  498.  1900. 

fjurgens.     Deutsche  Med.  Wochenschr.     XXIX.,  No.  47.  p.  1713. 

JFord.  The  classification  and  distribution  of  the  intestinal  bacteria  in  man. 
Studies  from  Rockefeller  Institute.  Vol.  II.  1904.  pp.  1-95. 


390  MANUAL    OF    BACTERIOLOGY. 

artificial  in  one  place  in  the  article  referred  to,  Ford  states  in  another  place, 
that  the  failure  to  agglutinate  with  dysenteric  serum  positively  differentiates  the 
B.  pseudodysentericus  from  B.  dysentera. 

Below  are  some  of  the  characteristics  noted  by  Ford.* 

Bacilli  measuring  0.5  by  1-2  microns  in  dimensions;  in  pairs  and  short  chains. 
Slow  motility  in  young  broth  and  in  agar  cultures,  motility  more  marked  in  older 
cultures.  Spores  absent.  On  agar  slant  growth  confined  to  the  line  of  inocu- 
lation. Deep  colonies  in  agar  round,  regular  and  opaque.  In  general  the 
colonies  resemble  those  of  B.  typhosus.  Growth  in  broth  abundant  with  heavy 
sediment,  but  no  pellicle.  On  gelatin  no  liquefaction.  Fennentation  in  dex- 
trose broth  grow  in  both  arms  of  the  tube,  reaction  in  closed  arm,  acid;  no  gas. 
Indol  produced  rarely  in  small  quantities.  It  is  found  in  the  lower  portions  of  the 
intestines,  but  also  occurs  in  the  stomach  and  duodenum  as  well,  in  persons 
not  affected  with  dysentery.  Does  not  agglutinate  with  the  serum  from 
persons  suffering  with  dysentery. 

Torreyf  summarizes  the  points  of  difference  between  the  true  dysentery 
bacillus  and  the  pseudo-forms  as  follows:  The  latter  spread  over  the  surface 
of  the  culture  plate  mere  than  the  B.  dysentericus,  .they  produce  finally  acidity 
in  litmus  milk,  do  not  absorb  agglutinins  of  B.  dysentericus.  The  realtionship 
between  the  B.  dysentericus  and  the  pseudo-forms  is  about  the  same  as  that 
existing  between  the  B.  diphtheria?  and  the  pseudo-diphtheria  bacilli. 


Spirillum  Cholerae  Asiaticae  (Comma  Bacillus  of  Cholera). 
— A  rod-shaped  organism,  with  rounded  ends.  It  is  usually 
curved,  hence  the  name  comma  bacillus  often  given  to  it;  but 
the  curve  is  often  very  slight.  The  curved  forms,  placed  end 
to  end,  may  produce  an  S-shaped  body.  The  length  is  from 
0.8  to  2  ft  and  the  breadth  from  0.3  to  0.4  /*.  In  cultures  some 
individuals  may  develop  into  genuine  spirilla.  In  the  whitish 
particles  found  in  the  stools  of  cases  of  cholera  the  organisms 
may  be  present  in  very  large  numbers.  In  these  particles  they 
may  exhibit  a  very  curious  arrangement,  lying  parallel  with 
one  another,  and,  as  remarked  by  Koch,  they  resemble  a 
school  of  fish  moving  up  stream.  Involution  forms,  irregular 
in  outline  and  staining  poorly,  are  often  seen  in  old  cultures. 
The  organism  is  motile,  having  a  flagellum  at  one  end.  It  does 
not  form  spores.  It  stains  with  the  ordinary  aniline  dyes,  but 
not  by  Gram's  method.  It  is  aerobic.  It  grows  at  the  room- 
temperature,  but  better  in  the  incubator.  On  the  ordinary 
media  the  growths  are  whitish.  It  grows  best  on  neutral  or 

*Loc,  cit. 

"fJourn.  Exper.  Med.     Vol.  VII.     1905.     pp.  365-400. 


PATHOGENIC    BACTERIA. 


391 


alkaline  media,  and  is  very  sensitive  to  a  small  amount  of  acid. 
It  liquefies  gelatin.  The  colonies  on  gelatin  plates  have  a 
very  characteristic  appearance.  They  are  nearly  round  at 
first,  and  granular  as  seen  under  the  low  power  of  the  micro- 
scope; but  at  the  end  of  about  twenty-four  hours  the  outline 
is  slightly  irregular,  and  the  surface  looks  as  though  it  were 
covered  with  finely  broken  glass.  The  outline  later  becomes 
still  more  irregular  or  scalloped.  As  liquefaction  of  the 
gelatin  takes  place  a  funnel-shaped  depression  is  formed,  into 
which  the  colony  sinks.  Gelatin  plates  should  be  kept  at  a 


FIG.  101. — Involution  forms  of  the  spirillum  of  cholera. — (Van  Ermengem.) 

temperature  of  from  20°  to  22°  C.  In  stab-cultures  in  gelatin 
a  white  growth  forms  around  the  stab,  and  at  the  end  of  about 
thirty-six  to  forty-eight  hours  a  funnel-shaped  depression 
occurs  on  the  surface,  owing  to  the  liquefaction  of  the  gelatin. 
This  depression  increases  in  size,  and  the  surface  of  the  lique- 
fied gelatin  seems  to  be  surrounded  by  an  air-bubble,  which 
appears  to  have  taken  the  place  of  the  part  of  the  fluid  gelatin 
which  has  evaporated.  In  the  deeper  portion  o  the  stab 
liquefaction  is  less  noticeable.  The  growths  on  agar  are  not 
characteristic.  In  bouillon  a  pellicle  forms  on  the  surface. 
On  potato  in  the  incubator  the  growth  is  whitish  or  brownish, 
not  conspicuously  elevated.  After  growing  it  in  Dunham's 


392 


MANUAL    OF    BACTERIOLOGY. 


peptone  solution  in  the  incubator  the  addition  of  sulphuric 
acid  develops  a  red  color,  owing  to  the  presence  of  indol  and 
nitrites — the  so-called  "cholera  red"  reaction.  Considerable 
doubt  has  recently  been  cast  upon  the  formation  of  nitrites  by 
the  cholera  spirillum.*  The  cholera-red  reaction  is  not 
confined  to  this  organism,  and  is  said  to  differ  from  the  nitroso- 
indol  reaction. 


FIG.  102. — Spirillum  of  cholera,  colonies  on  gelatin  plates.     (X  100  to  150.) 

(Frankel  and  P/eiffer.) 
a.  Twenty-four  hours  old.     b.  Thirty  hours  old.     c.  Forty-eight  hours  old. 


The  spirillum  of  cholera  is  said  to  be  very  sensitive  to  dry- 
ing, and,  provided  the  drying  be  complete,  is  usually  killed 
within  twenty-four  hours.  It  is  killedf  immediately  by  the 
temperature  of  boiling  water,  inj$  minutes  with  certainty  at 
80°  C.,  in  one  hour  at  56°  C.  It  may  retain  its  vitality  in  water 
for  a  long  time;  observations  vary  widely  in  respect  to  deter- 

*  Wherry.     Journal  of  Infectious  Diseases.  Vol.  II.     No.  3.     June  24,  1905. 
fKolle  in  Kolle  and  Wassermann.     Vol.  III.     1903.     p.  23. 


PATHOGENIC    BACTERIA. 


393 


mining  how  long.  In  the  ordinary  food-substances  it  may 
survive  long  enough  to  allow  them  to  act  as  carriers  of  the 
infection  if  eaten  raw.  It  is  an  important  fact  that  the  cholera 
spirillum  is  not  a  strict  parasite,  but  under  favorable  con- 
ditions it  may  maintain  its  vitality  for  some  time  outside  of 
the  human  body. 

The  animals  ordinarily  used  for  laboratory 
experiments  are,  in  their  normal  condition, 
not  susceptible  to  infection  with  the  spirillum 
of  cholera  through  the  alimentary  canal,  and 
no  animal  is  known  which  suffers  from 
spontaneous  cholera  excepting  man,  though  a 
disease  resembling  cholera  can  be  reproduced 
in  animals  when  certain  conditions  are  com- 
plied with.  The  acid  of  the  gastric  juice  de- 
stroys the  organism,  and  this  makes  it  im- 
possible to  infect  animals  by.  way  of  the 
alimentary  tract  unless  this  acidity  is  overcome 
with  an  alkali  before  the  introduction  of  the 

rilulirp  FIG.    103.— Spiril- 

lum    of     cholera, 

The  following  plan  was  adopted  by  Koch :  stab  -  culture  i  n 
The  gastric  juice  was  neutralized  with  a 
solution  of  sodium  carbonate;  the  movements 
of  the  intestines  were  quieted  by  the  injection 
of  i  c.c.  of  tincture  of  opium  for  each  200  grams  of  the  body- 
weight;  and  a  portion  of  pure  culture  of  the  cholera  spirillum 
was  introduced  into  the  stomach.  When  guinea-pigs  are 
treated  in  this  manner,  in  most  cases  a  condition  closely 
simulating  cholera  is  produced.  The  animal  dies  with 
symptoms  of  collapse.  The  small  intestine  is  more  or  less 
filled  with  a  watery,  flocculent  fluid  containing  a  large  number 
of  the  spirilla  of  cholera.  The  mucous  membrane  of  the 
intestine  is  swollen  and  reddened. 

When  guinea-pigs  receive  an  intraperitoneal  injection  fiom 


gelatin,  two  days 
old.  (Friinkelcnd 
Pfeiffer.) 


394  MANUAL    OF    BACTERIOLOGY. 

a  pure  culture,  death  usually  results,  apparently  from  the  toxic 
substances  contained  in  the  culture.  Pfeiffer  was  the  first  to 
show  that  an  animal  may  be  made  immune  from  cholera  by 
repeated  small  doses  of  cultures  which  have  been  heated 
in  order  to  kill  the  organism.  He  also  showed,  in  the  same 
connection,  that  when  living  comma  bacilli  are  introduced 
in  the  peritoneum  of  an  immune  animal  they  first  clump 
together  and  are  then  rapidly  destroyed  and  disintegrated 
(see  page  228);  furthermore,  that  a  drop  of  the  peritoneal 
fluid  added  to  a  hanging-drop  culture  of  the  cholera  spirillum 
produces  the  same  effect.  This  is  now  called  Pfeiffer's  phe- 
nomenon, and  is  the  underlying  principle  of  all  agglutination 
reactions,  such  as  the  Gruber-Widal  typhoid  test. 

It  seems  probable,  from  the  results  so  far  obtained,  that  it  is  practicable  'to 
use  injections  of  dead  cultures  upon  human  beings  with  safety,  and  in  this 
way  so  protect  healthy  persons  from  cholera  during  an  epidemic.* 

Haffkinef  elaborated  a  special  method  which  goes  by  his  name.  He  found 
that  by  passage  through  guinea-pigs  the  virulence  of  the  culture  could 
be  exalted  to  a  constant  maximum  point  which  he  calls  his  "virus  fixe."  This  is 
attained  by  a  passage  through  20  or  30  guinea  pigs.  The  guinea-pigs  are  all 
injected  into  the  peritoneal  cavity,  starting  by  inoculating  a  guinea-pig  with 
a  dose  larger  than  is  necessary  to  kill  the  animal,  and  inoculating  the  subsequent 
animals  of  the  series  with  the  peritoneal  exudate  of  the  dead  animals.  The 
exudate  must  be  exposed  to  the  air  for  15  hours  before  it  is  injected  into  the 
next  guinea-pig,  otherwise  it  is  found  to  be  not  so  virulent,  this  exposure  con- 
trary to  usual  experience  enhancing  the  virulence.  Small  guinea-pigs  give  less 
abundant  but  more  concentrated  exudate.  This  so-called  "virus  fixe"  is 
cultivated  in  beef-broth  at  39°  C.  in  a  current  of  air,  and  kept  in  this  manner 
until  cultures  made  daily  on  agar  fail  to  show  any  growth.  The  last  culture 
which  grows  injected  into  a  guinea-pig  subcutaneously  does  not  produce  any 
necrosis,  and  it  also  prevents  the  necrosis  of  the  skin  produced  by  "virus  fixe" 
when  so  injected.  Haffkine  inoculated  himself  with  the  attenuated  culture 
described,  and  6  days  later  with  the  "virus  fixe;"  he  then  proceeded  to  inject  a 
large  number  of  persons,  and  finally  to  try  its  inoculations  upon  more  than 
100,000  persons  in  localities  in  India,  with  apparently  somewhat  favorable  re- 
sults. The  inoculation  does  not  modify  the  course  of  the  disease  in  those  cases 
in  which  an  inoculated  person  contracts  the  disease.  The  percentage  of  death 
is  as  great  among  such  persons  as  among  uninoculated  persons  who  contract 
the  disease. 

Although  a  positive  demonstration  that  the  spirillum  of 
Koch  is  the  cause  of  cholera  is  lacking,  as  far  as  the  exact 

*Strong.     American  Medicine.     August  15,  1903. 

•fBull.  de  VInst.  Past.  Vol.  IV.,  Nos.  17  and  18.  1906.  pp.  697-705,  and 
737-747- 


PATHOGENIC    BACTERIA.  395 

reproduction  of  the  disease  in  animals  is  concerned,  the  neces- 
sary proof  has  been  supplied  by  the  accidental  or  intentional 
infection  of  laboratory  investigators  who  were  working  with 
cholera,  which  has  happened  on  several  occasions. 

Bacteriological  investigations  have  shown  that  the  spirilla  of 
cholera  are  present  in  very  large  numbers  in  the  watery  contents 
of  the  intestine,  especially  early  in  the  disease.  They  appear 
in  the  lumina  of  the  glands,  and  they  may  be  seen  underneath 
the  epithelial  cells.  They  may  occu'r  in  the  matters  vomited. 
They  have  been  found  in  the  feces  of  apparently  well  persons 
in  times  of  cholera  epidemic.  These  persons  may  spread 
the  disease,  and  are  to  be  regarded  as  bacillus  carriers  just  as 
in  the  case  of  typhoid  fever  where  healthy  persons  harbor 
the  bacilli  and  evacuate  them  in  the  stools.  They  usually 
are  not  found  widely  spread  through  the  organs  of  the  body. 
It  is  probable  that  the  symptoms  of  the  disease  result  from 
poisonous  substances  produced  by  the  spirilla  or  contained 
in  them. 

The  portal  of  entry  in  cholera  is  probably  always  the  ali- 
mentary tract,  and  the  infectious  agent  is  usually,  though  not 
always,  transmitted  through  drinking-water,  and  numerous 
epidemics  have  been  traced  to  this  source.  In  some  cases  the 
origin  of  the  contamination  of  the  water  with  cholera  dejecta 
has  been  demonstrated.  The  organism  may,  however,  be 
introduced  into  the  alimentary  tract  upon  any  and  every 
article  of  food.  It  may  be  conveyed  from  place  to  place  upon 
soiled  clothing  and  bedding,  and  then  be  brought  in  contact 
with  food.  Flies  also  probably  convey  the  organisms  from 
cholera  stools  to  articles  of  food.  In  order  to  combat  the 
spread  of  the  disease  the  excreta  and  bedding  should  be 
thoroughly  sterilized;  the  hands  of  the  attendants  should  be 
carefully  disinfected  and  all  food  should  be  cooked.  Although 
commoner  in  the  summer-time,  epidemics  of  cholera  have 
been  known  to  occur  in  the  winter. 


396  MANUAL    OF    BACTERIOLOGY. 

Bacteriological  Diagnosis  of  Cholera. — When  cases  sus- 
pected of  being  cholera  appear  in  a  community,  it  becomes 
a  matter  of  the  utmost  importance  to  determine  the  exact 
nature  of  the  disease  in  order  that  it  may  not  become  epidemic. 
One  of  the  first  occasions  when  bacteriological  methods  were 
put  into  practice  in  the  diagnosis  of  cholera  was  at  the  time 
of  the  appearance  of  that  disease  in  the  port  of  New  York  in 
1887. 

According  to  Koch,  the  diagnosis  may  be  made  in  twenty- 
four  hours  or  less.  It  is  important  to  obtain  the  discharges 
from  the  intestines  as  early  in  the  course  of  the  disease  as 
possible,  and  while  they  are  perfectly  fresh.  It  may  be  neces- 
sary, however,  to  examine  the  moist  dejecta  on  the  linen  or 
clothing,  when  no  other  material  is  available. 

In  the  first  place,  one  of  the  small,  partly  solid  particles 
which  may  be  found  in  the  discharges  from  the  intestines 
should  be  smeared  upon  a  cover-glass,  fixed  in  the  usual 
manner,  stained  with  one  of  the  aniline  dyes,  and  examined 
with  the  microscope.  If  taken  early  in  the  disease,  the  comma 
bacilli  may  be  present  in  large  numbers,  and  they  are  likely 
to  be  arranged  in  parallel  groups,  as  already  described.  If 
comma-shaped  bacilli  are  thus  found,  a  strong  probability 
is  created  that  the  disease  is  Asiatic  cholera.  The  motility  of 
the  organisms  can  be  determined  by  examination  in  the  hang- 
ing-drop. It  is  to  be  remembered  that  spirilla  of  various 
forms  are  common  in  the  normal  mouth,  and  may  appear  in 
the  stools  (see  pages  161  and  274). 

The  method  of  procedure  recommended  by  Koch,  Kirchner 
and  Kolle,*  sent  out  by  the  German  minister  of  medical  affairs 
to  the  different  directors  of  hygienic  institutes  in  Germany 
for  their  guidance  is  in  substance  as  follows: 

Microscopic  examination  of  the  stools,  preferably  of  the 
mucous  flakes  should  be  made  by  smearing  the  material  over 

*Kolle  and  Wassermann.  Bd.  III.     1903.     p.  42  et  seq. 


PATHOGENIC    BACTERIA.  397 

the  cover-glass  in  the  usual  manner,  and  staining  with  carbol- 
fuchsin  diluted  i  to  9.  Also  microscopic  examination  of 
hanging  drops  prepared  in  peptone  solution.  These  prep- 
arations are  made  and  examined  immediately  and  after  30 
minutes  in  the  incubator  at  37°  C.,  also  dried  and  stained. 
The  peptone  solution  is  described  below. 

Gelatin  plates  should  also  be  made  from  the  same  material. 
These  should  be  made  in  2  series  of  3  plates  each.  The 
first  tube  of  melted  gelatin  is  inoculated  with  a  loopful  of  the 
stool,  preferably  from  a  mucous  flake.  The  other  tubes  are 
inoculated  in  the  customary  manner,  3  loops  from  the  first  to 
the  second,  3  loops  from  the  second  to  the  third.  The  plates 
should  be  placed  at  22°  C.  for  18  hours  and  then  examined 
with  the  low  power  of  the  microscope.  Impression  preparations 
and  smears  should  also  be  examined,  and  pure  culture  made 
from  colonies. 

Agar  plates  should  also  be  made  in  the  manner  described 
for  gelatin  plates,  or  the  loop  of  material  from  the  stool  may  be 
stirred  up  in  5  c.c.  of  bouillon,  and  three  plates  made  from 
this  suspension,  using  one  loopful  for  each  plate. 

Forthermore,  6  tubes,  each  with  10  c.c.  of  the  peptone 
solution  described  below,  are  inoculated  each  with  i  loopful 
of  the  feces,  and  examined  with  the  microscope  after  6  to  12 
hours  in  the  incubator  at  37°  C.  without  shaking  up  the  con- 
tents of  the  tube.  The  tube  showing  organisms  most  resem- 
bling cholera  is  used  for  the  purpose  of  making  plates  in  agar 
and  in  gelatine  as  described  above  for  plates  directly  from 
the  stool. 

'  In  addition  to  the  plates,  3  tubes  of  peptone  are  inoculated, 
each  with  i  loopful.  The  peptone  tubes  should  be  warmed  to 
37°  C.  before  they  are  inoculated. 

Lastly,  a  flask  containing  50  c.c.  of  the  peptone  solution  is 
inoculated  with  i  gram  of  the  stool,  and  examined  of  6  to.  12 
hours  as  above. 


398  MANUAL    OF    BACTERIOLOGY. 

The  peptone  solution  is  made  by  taking  i  part  of  the 
enriching  fluid  described  below  for  the  examination  of  water 
for  the  cholera  spirillum  (p.  399),  and  diluting  with  9  parts  of 
water,  distributing  it  in  test-tubes,  10  c.c.  in  each,  and 
sterilizing. 

At  the  time  that  the  first  smear  preparations  and  gelatin 
plates  are  prepared,  tubes  of  peptone  solution  should  be 
inoculated  directly  from  the  intestinal  contents,  and  kept  in 
the  incubator  (Schottelius).  After  development  has  occurred, 
the  production  of  indol  may  be  tested  by  the  addition  of  sul- 
phuric acid.  These  tubes  are  especially  valuable  when  un- 
favorable material  or  when  material  containing  small  numbers 
of  the  spirilla  is  used.  In  the  incubator  the  spirilla  may  be 
expected  to  multiply  in  the  peptone  solution  rapidly,  and  to 
appear  upon  the  surface  of  the  liquid  in  large  numbers,  even 
forming  a  visible  film  in  six  hours.  Smears  may  be  made 
from  the  surface  part  of  these  tubes,  stained,  and  examined 
with  a  microscope.  From  the  same  material  gelatin  plates 
should  be  prepared,  and  examined  as  soon  as  the  colonies 
develop. 

Ashburn  and  Craig*  have  sounded  a  warning  against  too 
great  reliance  upon  bacteriological  results  in  the  early  diag- 
nosis of  cholera.  It  is  seldom  that  even  approximately  pure 
cultures  of  the  cholera  bacillus  are  obtained  from  stools,  and 
from  mixed  cultures  from  the  stools  they  obtained  the  "  cholera- 
red"  reaction  in  one  case  only.  The  reaction  can  usually  be 
obtained  in  pure  cultures  by  using  media  prepared  with 
Grubler's  peptone,  but  not  with  other  peptones. 

When  cultures  are  obtained,  their  effects  may  be  tested 
upon  guinea-pigs,  by  injecting  them  into  the  peritoneum. 

The  production  of  Pfeiffer's  phenomenon  is  at  present 
regarded  as  the  most  important  and  final  means  of  diagnosis 
between  the  cholera  spirillum  and  related  forms.  This  con- 

*Journ.  Am.  Med.  Assn.  Vol.  XL VIII.     No.  8.     Feb.  23,  1907.     p.  692. 


PATHOGENIC    BACTERIA.  399 

sists  in  testing  the  suspected  organism  with  serum  from  an 
animal  immunized  with  cultures  of  cholera  bacilli,  as  already 
explained  above. 

The  serum  employed  in  this  test  must  of  course  be  prepared 
by  injecting  the  animal  furnishing  the  serum  with  pure  cultures 
of  a  strain  of  undoubted  origin.  The  potency  of  the  serum 
itself  must  be  determined  by  trial  upon  cultures  of  known 
identity.  This  is  done*  by  preparing  a  series  of  dilutions  of 
the  serum  with  normal  salt  solution  in  test- tubes,  each  test-tube 
to  contain  i  c.c.  of  the  diluted  serum.  To  each  of  the  series 
of  tubes  one  loopful  from  an  eighteen-hour  agar  culture  is 
added,  and  the  tubes  so  prepared  are  placed  in  the  incubator 
at  37°  C.  for  one  hour.  The  strength  of  the  serum  is  denoted 
by  the  dilution  which  causes  an  agglutination  which  can  be 
seen  with  the  naked  eye.  Thus  a  serum  of  the  strength 
i-iooo  is  such  that  i  c.c.  of  a  dilution  of  i  part  of  serum  to 
999  parts  of  normal  salt  solution  causes  a  visible  clumping 
of  i  loopful  of  the  cholera  spirillum  taken  from  an  18  hour 
agar  culture.  If  the  strength  of  the  serum  is  thus  determined, 
and  if  a  culture  obtained  from  the  stool  of  a  person  presenting 
suspicious  symptoms  of  cholera,  it  is  safe  to  make  a  positive 
diagnosis. 

For  examining  suspected  water  for  the  spirillum  of  cholera 
Kollej  advises  the  following  method:  An  enriching  fluid 
is  first  prepared  which  consists  of  i  liter  of  distilled  water 
in  which  are  dissolved  by  warming  100  grams  Witte's  peptone, 
100  grams  salt,  i  gram  potassium  nitrate,  and  2  grams  crys- 
tallized carbonate  of  sodium.  The  solution  is  filtered,  and 
distributed  into  flasks,  100  c.c.  in  each  flask,  and  sterilized. 

At  least  i  liter  of  the  water  to  be  examined  is  taken,  and  to  it 
is  added  the  contents — 100  c.c. — of  one  of  the  flasks  of  enrich- 
ing fluid.  It  is  then  distributed  into  flasks,  100  c.c.  in  each 

*Kolle  and  Wassermann.     Bd.  III.  1903.  p.  39. 

fKolle  and  Wassermann's  Handbuch.  Vol.  III.  1903.  pp.  44~45- 


400  MANUAL    OF    BACTERIOLOGY. 

flask,  and  put  in  the  incubator  at  37°  C.  and  examined  in  from 
8  to  18  hours.  The  examination  consists  in  the  microscopic 
examination  of  the  scum  which  forms  more  or  less  on  the  top 
of  the  fluid — the  flask  showing  the  most  scum  is  selected— 
and  in  the  application  of  the  various  tests  for  the  identification 
of  the  organism  given  above.  See  also  page  145. 

Since  Koch's  discovery  of  the  cholera  spirillum  in  1883-84 
a  considerable  number  of  bacteria  have  been  described  which 
resemble  the  cholera  spirillum  more  or  less  closely,  and  these 
have  to  be  taken  into  account  in  making  examinations  of 
suspected  material  of  any  sort.  This  is  particularly  necessary 
in  the  investigation  of  water,  in  which  such  cholera-like  spirilla 
seem  to  occur  quite  frequently. 

Vibrio  Metchnikovii. — A  comma-shaped  organism,  which, 
though  somewhat  shorter  and  thicker  than  the  cholera  bacillus, 
is  very  similar  to  the  latter  in  form,  and,  like  this,  may  some- 
times form  genuine  spirilla.  It  is  motile  and  has  a  flagellum 
at  one  end.  It  does  not  form  spores.  It  is  aerobic.  It  stains 
with  the  aniline  dyes,  and  is  not  stained  by  Gram's  method. 
It  grows  at  the  room-temperature.  It  liquefies  gelatin  some- 
what more  rapidly  than  the  spirillum  of  cholera.  The  colonies 
on  gelatin  plates  are  not  all  alike;  some  of  them  resemble 
those  of  Vibrio  proteus,  and  others  are  extremely  like  those  of 
the  spirillum  of  cholera.  It  grows  upon  the  usual  media. 
Coagulated  blood-serum  is  liquefied  by  it.  The  growth  on 
agar  is  grayish  to  yellowish,  and  abundant.  It  forms  a  pellicle 
on  bouillon.  In  milk  an  acid  reaction  is  developed  with  coagu- 
lation. In  peptone  solution  it  produces  indol  and  nitrates  like 
the  spirillum  of  cholera.  It  is  said  to  give  the  nitrosoindol 
reaction  more  intensely  than  the  spirillum  of  cholera. 

It  was  discovered  in  chickens  suffering  from  gastro-enteritis. 
It  is  pathogenic  for  chickens,  pigeons  and  guinea-pigs;  less  so 
for  mice  and  not  at  all  for  rabbits.*  The  comma-shaped 

*Giinther.    Loc.  clt.     p.  683. 


PATHOGENIC    BACTERIA.  40 1 

organisms  are  found  in  the  blood  in  guinea-pigs,  pigeons 
and  young  chickens. 

Vibrio  Proteus  (Finkler  and  Prior). — A  comma-shaped 
organism  somewhat  larger  than  the  spirillum  of  cholera,  some- 
times exhibiting  genuine  spiral  forms,  and  also,  at  times, 
involution  forms.  It  is  motile  and  has  a  flagellum  at  one  end. 

The  developments  of  the  colonies  in  gelatin  and  the  lique- 
faction of  this  medium  are  more  rapid  than  with  the  cholera 

'  V'  HP" 

-         >  ~_ 

** 
V 

-.  -          ~  *•  ~ 

*" 

$f~    *  *  S      ' 

F  -T          ,  -* 


f 


,         ( 

•00. 

f*  * 

FIG.  104. — Vibrio  proteus.* 

spirillum.  At  the  end  of  twenty-four  hours  the  colonies  are 
all  circular,  larger  than  those  of  the  spirillum  of  cholera,  and 
uniformly  granular  when  slightly  magnified.  On  the  other 
culture-media  the  growths  are  usually  whitish.  On  potato  it 
produces  an  abundant,  moist,  grayish-yellow  deposit,  and 
grows  at  the  room-temperature.  It  liquefies'  coagulated  blood- 
serum;  milk  becomes  acid.  In  peptone  solution  is  does  not 
usually  form  indol,  but  occasionally  it  does  so.  It  is  less 
pathogenic  to  animals  than  the  spirillum  of  cholera.  It  was 

*  The  magnification  is  a  little  greater  than  in  the  other  photomicrographs. 
26 


402  MANUAL    OF   BACTERIOLOGY. 

supposed    by  its    discoverers    to   be    the    cause    of    cholera 
nostras,  but  it  appears  to  have  no  relation  to  that  disease. 

Spirillum  Millieri. — A  comma-shaped  organism  resem- 
bling Vibrio  proteus  in  many  respects,  and  probably  identical 
with  it.  In  gelatin  it  grows  more  rapidly,  and  produces  lique- 
faction more  rapidly  than  the  spirillum  of  cholera.  On  gela- 
tin plates,  at  the  end  of  twenty-four  hours,  the  colonies  are 
uniformly  circular  and  granular,  lying  in  little  depressions 
resulting  from  the  liquefaction  of  the  gelatin.  Its  growths 
in  the  other  media  are  not  characteristic.  It  liquefies  blood- 
serum.  It  does  not  produce  indol  as  a  rule.  It  is  less  toxic 
to  animals  than  the  spirillum  of  cholera.  It  was  isolated  by 
Miller  from  a  carious  tooth. 

See  also  Spirillum  sputigenum,  Part  III. 

Spirillum  Tyrogenum  (Deneke). — A  comma-shaped  organ- 
ism, not  so  large  as  the  spirillum  of  cholera.  It  is  motile, 
having  a  flagellum  at  one  end.  It  does  not  form  spores.  In 
cultures,  genuine  spirilla  may  develop.  Gelatin  is  liquefied 
more  rapidly  than  by  the  spirillum  of  cholera,  and  the  colonies 
develop  more  rapidly.  The  circumference  of  the  colony  is 
round,  the  surface  may  appear  somewhat  granular,  and  it  has 
a  greenish-brown  color,  seen  under  the  low  power.  Milk  con- 
taining litmus  becomes  acid,  is  subsequently  decolorized,  and 
is  also  coagulated.  It  liquefies  coagulated  blood-serum.  It 
does  not  form  indol  in  Dunham's  peptone  solution  as  a  rule. 
No  pellicle  forms  in  cultures  upon  bouillon.  It  is  less  toxic 
to  animals  than  the  spirillum  of  cholera.  It  was  isolated 
originally  from  old  cheese. 

Vibrio  Berolinensis. — A  comma-shaped  organism  resem- 
bling the  spirillum  of  cholera  in  form  and  in  the  position  of  its  I 
flagellum.     It  does  not  stain  by  Gram's  method.     It  grows  at  ! 
the  room-temperature,  but   more   rapidly  in   the  incubator,  i 
The  colonies  upon  gelatin,  one  or  two  days  old,  when  magnified, 
are  decidedly  more  finely  granular  and  more  transparent  than  ; 


PATHOGENIC    BACTERIA.  403 

those  of  the  spirillum  of  cholera,  and  the  margin  is  almost 
absolutely  smooth  and  circular.  As  the  colonies  become  older 
they  assume  a  more  irregular  and  lobulated  appearance,  but 
are  still  more  finely  granular  than  the  colonies  of  the  cholera 
spirillum.  Gelatin  is  very  slowly  liquefied.  Its  growth  on  the 
other  culture-media  is  not  remarkable.  It  forms  indol  in  pep- 
tone solution,  and  it  increases  in  the  upper  layers  of  the  fluid. 


FIG.  105. — Spirillum  of  relapsing  fever  in  the  blood.     Sketched 
from  a  stained  specimen. 

When  guinea-pigs  are  inoculated  in  the  peritoneal  cavity,  death 
occurs  in  one  to  two  days.  This  organism  was  discovered  in 
the  water-supply  of  Berlin. 

Other  spirilla  have  been  isolated  rom  water  by  Giinther 
(Vibrio  aquatilis  in  Spree  water) ;  by  Dunbar  from  the  Elbe 
River;  by  Russell  from  the  Gulf  of  Naples;  by  Heider  from  the 
water  of  the  Danube  Canal;  and  in  America,  by  Abbott,  from 
the  water  of  the  Schuylkill  (Vibrio  Schuylkiliensis) ;  and  many 
others  have  been  described  to  which  the  limits  of  this  work 
will  not  permit  of  further  allusion. 


404  MANUAL    OF    BACTERIOLOGY. 

Two  vibrios,  V.  Massaua  and  V.  Ghinda,  were  isolated  in 
the  places  after  which  they  are  respectively  called  by  Pasquale.* 
Both  were  held  to  be  genuine  cholera  spirilla  at  first,  but  the 
failure  to  agglutinate  with  cholera-immune  serum  differentiates 
them  from  this  organism.  V.  Massaua  was  isolated  from  the 
dejecta  of  a  patient  who  was  suffering  from  some  other  disease 
than  cholera,  though  there  had  been  an  outbreak  of  cholera 
at  the  place  before  Pasquale's  arrival.  The  other  vibrio  was 
isolated  from  drinking  water.  Gotschlich  21  cultures  of 
vibrios  from  various  material  in  Alexandria,  in  Egypt,  all  of 
which  could  be  differentiated  from  cholera  by  means  of 
immune  serum,  f 

The  Spirillum  or  Spirochaeta  Obermeieri  (of  Relapsing 
Fever). — A  slim  spirillum  with  numerous  turns,  1 6  to  40  /*  in 
length.  The  ends  are  pointed.  It  is  actively  motile.  The 
spirillum  is  not  stained  by  Gram's  method,  but  may  be  colored 
by  the  ordinary  aniline  dyes.  The  organism  has  never  been 
cultivated.  It  is  found  abundantly  in  the  blood  and  in  the 
spleen  during  the  attack  of  fever.  The  spleen  is  enlarged. 
The  disease  has  been  produced  in  apes  by  inoculating  them 
with  blood  taken  from  men  having  the  disease. 

Novy  and  KnappJ  came  to  the  conclusion  as  the  result  of 
their  investigations  in  this  direction  that  the  S.  obermeieri 
is  to  be  classed  with  the  bacteria  and  not  with  the  protozoa. 
They  also  found  that  besides  human  beings,  monkeys,  white 
mice,  rats;  wild  and  tame  are  subject  to  infection  with  the 
organism;  moreover,  monkeys,  mice  and  rats  can  be  promptly 
cured  by  injections  of  hyperimmunized  blood.  S.  obermeieri 
can  be  made  to  pass  through  a  Berkefeld  filter. 

In  a  preliminary  note  Novy  and  Knapp§  report  the  culti- 
vation of  a  spirillum  which  they  call  S.  obermeieri  with  reserve; 

*  Kolle  and  Wassermann.  Bd.  III.     1903.     p.  71. 

"flbid.  p.  72. 

\Journ.     Infec.     Diseases.  Vol.  III.     1906.     pp.  291-393. 

\Journ.     Am.  Med.  Assn.  No.  26.  Vol.  XLVII.     Dec.  29,  1906. 


PATHOGENIC    BACTERIA.  405 

for  although  it  was  obtained  from  a  case  of  relapsing  fever,  they 
have  grounds  for  the  belief  that  the  organism  causing  the  disease 
in  America  differs  from  that  found  in  the  eastern  disease.  The 
method  employed  consists  in  the  use  of  collodion  sacs  filled 
with  rats'  blood.  These  sacs  are  inoculated  with  a  small 
amount  of  blood  containing  the  spirillum,  and  are  then  placed 
in  the  peritoneal  cavity  of  white  rats. 

Spirochaeta  Pallida. — First  observed  by  Schaudinn  and 
Hoffmann*  in  recent  as  well  as  more  advanced  syphilitic 
lesions,  on  the  surface  and  deep  in  the  tissues  in  chancres, 
indolent  buboes  and  papules.  These  observations  have  been 
abundantly  corroborated,  but  for  diagnostic  purposes  they  can 
scarcely  yet  be  employed  with  certainty  on  account  of  the 
difficulty  of  recognizing  the  spirochaeta  in  exposed  lesions  such 
as  mucous  patches. 

Flexner|  finds  that  silver  impregnation  often  brings  out 
many  more  spirals  than  the  anilin  dyes.  He  furthermore 
finds  that  unexplained  factors  affect  the  stain  with  anilin  dyes, 
and  that  the  best  results  are  to  be  obtained  with  Stern'sJ 
method  of  staining  with  silver. 

It  is  asserted  that  the  spirillum  is  transferred  by  bed-bugs 
from  one  person  to  another.  § 

Grouven||  succeeded  in  producing  a  disease  with  lesions 
characteristic  of  syphilis  in  a  rabbit  by  intraocular  inoculation. 
S.  pallida  was  demonstrated  in  some  of  the  lesions  in  the 
rabbit. 

Levaditi  and  Mclntosh  *[[ '  claims  to  have  cultivated  an  or- 
ganism identical  with  S.  pallida.  in  all  respects,  except  that  it  was 

*Schaudinn  and  Hoffmann.  Vorlaufiger  Bericht  iiber  das  Vorkommen 
von  Spirochaeten  in  syphililischen  Krankheitsprodukten  und  bei  Papillomen 
Arbeiten  aus  dem  Kaiserhschen  Gesundheitsamt.  Bd.  XXII.,  p.  527,  1905. 

•\Journ.  Exper.  Medicine.     Vol.  IX.     1907.     pp.  464-472. 

\Berl.  klfn.  Wochenschr.     XLIV.     1907.     p.  400. 

§Karlinski.     Centralblatt  }iir  Bakteriologle.     Bd.  XXXI.     Original.     1901 

\\Deutsche  Med.  Wochenschr.     Nov.  21,  1907. 

\Ann.  de  I'Inst.  Past.     Vol.  XXI.,  No.  10.     Oct.  25,  1907.     p.  784. 


406  MANUAL    OF    BACTERIOLOGY. 

non-pathogenic,  by  the  use  of  collodion  sacs  placed  in  the  peri- 
toneal cavity  of  apes.  Metchnikoff  and  Roux  found  the  same 
organism  in  the  monkeys  which  they  had  successfully  inocu- 
lated with  syphilis. 

The  evidence  is  accumulating  rapidly  in  favor  of  this 
organism  as  the  cause  of  syphilis.*  It  is  4  to  14  /*  long, 
J/*  thick  and  has  6  to  14  turns.  It  is  actively  motile.  Stained 
with  great  -difficulty.  The  following  stain  was  recommended 
originally,  and  more  recently  a  variety  of  stains  have  also  been 
employed  by  different  observers: 

(1)  Three  parts  Giemsa's  eosin  solution  (2.5  c.c.  i  per  cent,  eosin  solution 
in  500  c.c.  water). 

(2)  Three  parts  asur  I  solution  (i  gram  asur  in  1000  c.c.  water). 

(3)  Three  parts  asur  II  solution  (0.8  gram  in  1000  water). 

Mix  and  stain  dried  cover-glass  preparations  from  16  to  24  hours;  wash, 
dry  and  mount  in  balsam. 

Spirochaeta  Refringens. — Found  less  frequently  than  S. 
pallida  in  the  same  locations  as  the  latter.  Is  larger  and 
stains  more  easily  than  S.  pallida. 

*Spirobacteria  in  the  Lesions  of  Syphilis.  Journal  o}  the  American  Medical 
Association.  Vol.  XLIV.,  No.  22.  p.  1790.  1905. 

Schultz.     Journ.     Med.  Res.     XV.  1906.     pp.  363-381. 


PATHOGENIC  PROTOZOA. 

PROTOZOA  are  unicellular  animal  organisms.  They  are 
attracting  ever  increasing  attention  since  they  have  been 
found  to  play  a  very  important  part  in  the  causation  of  dis- 
eases, particularly  of  diseases  peculiar  to  tropical  countries, 
and  as  they  are  studied  by  methods  that  have  much  in  common 
with  those  used  for  the  bacteria  they  may  be  considered  here 
briefly.  Protozoa  are  numerous  in  pond  and  ditch  water, 
and  these  species  seem  to  be  harmless.  However,  many 
diseases  of  the  lower  animals  are  caused  by  protozoa,  such  as 
surra,  Texas  fever  and  coccidium  disease  of  rabbits.  Birds,* 
reptiles  and  frogsj  may  show  organisms  in  the  blood  resem- 
bling the  parasites  o£  malaria.  Until  recently  it  has  been 
doubtful  whether  any  pathogenic  protozpon  has  ever  been 
propagated  in  pure  culture  outside  of  the  body  of  the  host. 
This  has  been  accomplished  by  Novy  and  MacNeal  for  a 
parasite  (Trypanosoma)  from  the  blood  of  the  ratj  and  from 
many  species  of  birds§  on  rabbit-blood-agar. 

Novy  1 1  gives  the  following  classification  of  the  pathogenic 
protozoa  which  are  of  most  interest:  Trypanosomata,  met 
free  in  the  blood  plasma;  hemocytozoa,  found  in  the  blood- 
cells,  represented  by  the  malarial  parasite  in  man  and  by 
related  organisms  in  the  lower  animals,  also  by  the  piro- 
plasmata  found  in  Texas  cattle  fever  and  allied  affections; 
amebae,  found  in  the  intestines  in  dysentery  and  elsewhere. 
Other  forms  of  pathogenic  protozoa  have  been  recognized, 

*Opie  and  MacCallum.     Journal  Experimental  Medicine.     Vol.  III. 

fLangmann.  New  York  Medical  Journal.  January  7,  1899.  Williams 
and  Lewis. 

tNovy  and  MacNeal.  Contributions  to  Medical  Research.  Dedicated  to 
Victor  C.  Vaughan.  1903. 

§Novy  and  MacNeal.  On  the  Trypanosomes  of  Birds.  Journal  of  In- 
fectious Diseases.  Vol.  II.,  No.  2.  p.  257.  March,  1905. 

HReprint  from  Journ.  Am.  Med.  Assn.     Jan.  5  and  i2;  1907.     Vol.  XLVIII. 

407 


408  MANUAL    OF    BACTERIOLOGY. 

but  these  are  as  yet  of  subsidiary  importance  compared  with 
those  just  named. 

Trypanosomes.* — The  trypanosomes  are  roughly  spindle- 
shaped  and  approximately  crescentic.  Each  cell  has  a  sharp 
posterior  extremity;  the  anterior  extremity  narrows  into  a 
single  long  flagellum  which  is  in  active  motion  during  life. 
An  undulating  membrane  surrounds  the  organism  from 
near  the  posterior  extremity  to  the  base  of  the  flagellum. 
This  membrane  can  be  seen  with  the  microscope  extending 
like  a  fin  on  either  side.  The  undulating  membrane  probably 
constitutes  the  organ  of  locomotion;  not  the  flagellum  as  in 
the  bacteria;  since  in  one  species  the  flagellum  is  rudimentary 
or  lacking  and  still  the  organism  is  endowed  with  the  power 
of  locomotion,  and,  moreover,  in  cultures  in  which  the  undulat- 
ing membrane  is  poorly  developed,  but  in  which  the  flagellum 
is  very  long  it  may  show  scarcely  any  motion.  The  cells  consist 
of  nearly  colorless,  almost  homogeneous  protoplasm.  When 
stained  by  the  Romanowsky  method  various  structures  are 
brought  out.  The  nucleus  which  is  near  the  anterior  end 
stains  blue.  The  flagellum  stains  deep  red.  A  body  known 
as  the  micro-nucleus  or  centrosome  or  blepharoplast  which  is  a 
prominent  object  near  the  posterior  end,  and  which  is  con- 
nected with  the  flagellum  by  a  distinct  line  passing  in  the 
undulating  membrane  along  the  side  of  the  organism,  in  fact 
it  is  continued  into  the  flagellum,  is  also  stained  deep  red. 
The  periblast  may  also  take  a  red  stain.  The  plasma  stains 
blue. 

The  adult  cell  is  25  mikrons  long  by  1.5  wide,  about  3.5 
times  as  long  and  about  one-fifth  as  wide  as  a  red  blood- 
corpuscle.  Multiplication  takes  place  by  obliquely  longitu- 
dinal division  only.  Transverse  division  has  never  been 

^Following  descriptions  of  trypanosomes  is  adapted  from  Novy.  LOG.  cit. 
For  very  full  account  of  trypanosomes  previous  to  1903,  see  Musgrave  and 
Clegg.  Bureau  of  Gov.  Labs.,  Philippine  Islands,  No.  5.  1903.  248  pages. 


PATHOGENIC    PROTOZOA.  409 

observed.  Conjugation  has  also  not  been  observed  at  least 
satisfactorily.  The  blepharoplast  is  the  first  to  begin  dividing, 
the  flagellum  remaining  attached  to  one  of  the  resulting  halves, 
while  a  new  flagellum  develops  on  the  other  half.  The  division 
of  the  nucleus  follows  next  in  order  as  a  rule,  though  this  may 
precede  the  division  of  the  blepharoplast.  The  active  division 
of  the  nuclei  and  plepharoplast  give  rise  to  rosette  formation 
owing  to  delayed  division  of  the  cell  protoplasm. 

Cultivation  of  Trypanosomes. — Many  trypanosomes  are 
much  alike  morphologically  so  that  they  are  hardly  dis- 
tinguishable with  the  microscope  alone,  but  in  cultures  the 
different  species  present  points  of  difference.  The  culture 
medium  adapted  to  their  growth  consists  of  equal  parts  of 
defibrinated  rabbit's  blood  serum  and  ordinary  nutrient 
agar.  The  method  of  preparation  of  the  medium  consists 
in  melting  up  tubes  of  agar  and  after  cooling  them  down  to 
50°  C.  adding  the  blood-serum.  The  tubes  are  allowed  to 
set  in  an  inclined  position,  then  stood  upright  to  allowed  the 
water  which  separates  out  to  accumulate  at  the  bottom  of  the 
incline.  Tubes  should  be  fresh,  since  it  is  necessary  to  have 
abundant  moisture  in  the  tube.  After  inoculation,  the  tubes 
are  kept  at  25°  C.  or  in  the  room.  First  growth  is  observed 
in  about  three  or  four  days  to  one  week.  Initial  cultures  are  not 
so  vigorous  as  subsequent  cultures.  Usually  there  is  no 
growth  visible  with  the  naked  eye,  but  sometimes  the  growth 
is  evdent  in  this  way.  Pure  growths  may  be  obtained  by 
making  streaks  on  blood-agar  plates.  The  forms  met  with 
in  cultures  differ  from  those  seen  in  the  blood  of  an  infected 
animal.  They  are  usually  smaller  in  the  cultures  than  in 
the  blood,  although  sometimes  they  are  much  longer  than 
these.  The  blepharoplast  is  usually,  though  not  always, 
lateral  or  anterior  to  the  nucleus  in  the  cultivated  forms. 
Some  species,  as  Tr.  avium,  show  two  distinct  forms  in  cultures 
giving  rise  to  the  suggestion  of  sexual  difference.  Trypano- 


410  MANUAL    OF    BACTERIOLOGY. 

somes  found  in  the  stomachs  of  mosquitoes,  tsetse  flies,  house 
flies,  etc.,  show  forms  like  those  met  with  in  cultures. 

From  accumulating  data  it  would  appear  likely  that  all 
species  of  animals  may  show  trypanosomes  in  the  blood. 
Novy  and  McNeal  were  the  first  to  cultivate  them  in  pure 
cultures.  Herbert  U.  Williams  and  Lewis  were  the  first  to 
cultivate  the  frog  trypanosome,  Tr.  rotatorium,  and  this 
furnishes  an  excellent  example  for  class  demonstration. 

Tr.  evansi  is  found  in  the  blood  of  animals  in  India  and  the  East,  generally 
suffering  from  a  disease  called  surra.  Horses,  mules,  camels,  dogs  and  cattle 
are  particularly  subject  to  the  disease  which  is  characterized  by  remittent  fever 
accompanied  by  anemia  and  wasting,  edema  of  the  legs,  belly  and  other  parts, 
with  a  discharge  from  eyes  and  nose.  The  organism  is  transferred  from  sick 
to  healthy  animals  through  the  agency  of  flies.  The  disease  is  seldom  fatal. 

Tr.  brucei,  almost  indistinguishable  from  Tr.  evansi,  causes  the  tsetse  fly  sick- 
ness, nagana,  of  Africa,  a  most  fatal  disease  of  horses,  donkeys,  dogs,  and 
cattle.  Nearly  all  mammals  are  susceptible  to  spontaneous  or  to  artificial  infec- 
tion. Man  appears  to  be  immune.  Tr.  brucei  was  the  first  pathogenic  try- 
panosome which  was  cultivated  artificially.  Differs  markedly  in  cultures  from 
Tr.  lewisi  and  Tr.  evansi. 

Tr.  equiperdum  causes  the  disease  of  horses,  known  as  dounne.  The  disease 
is  not  confined  to  tropical  countries,  but  is  found  in  many  parts  of  Europe  as 
well.  It  is  said  also  to  be  met  with  in  America.  It  is  not  spread  as  other 
diseases  which  are  caused  by  trypanosomes  through  the  agency  of  flies;  but 
only  by  sexual  contact,  and  hence  is  called  "mal  du  coit,"  it  is  also  called  horse 
syphilis  owing  to  suggestive  skin  lesions.  The  diseases  may  be  communi- 
cated to  asses,  dogs  and  rabbits.  Rats,  mice  and  guinea-pigs  are  refractory. 

Tr.equinum  causes  the  disease  known  as  "mal  de  caderas,"  occurring  almost 

,  exclusively  in  horses.     It  is  characterized  by  remittent  fever,  rapid  loss  in  weight, 

with  eventually  paralysis  of  the  hind  quarters.     The  organism  has  no  blepharo- 

plast  which  distinguishes  it  from  all  other  trypanosomes.     No  definite    success 

has  attended  efforts  at  cultivation  of  Tr.  equinum. 

Tr.  dimorphon  causes  a  disease  of  horses  in  Senegambia.  No  other  domestic 
animal  than  the  horse  suffers  spontaneously,  but  most  mammals  can  be  arti- 
ficially infected. 

Tr.  theileri  causes  a  disease  of  cattle  in  South  Africa. 

Tr.  gambiensis  is  the  cause  of  the  sleeping  sickness  of  West  Africa.  The 
disease  is  spread  through  the  agency  of  the  tsetse  fly,  though  a  different  species 
of  this  fly  from  that  which  spreads  the  nagana  of  South  Africa.  The  disease 
is  characterized  by  two  stages:  A  mild  stage  marked  by  mild  symptoms 
consisting  of  some  fever,  slight  edema  and  erythema;  the  second  stage  is  the 
sleeping  stage  always  terminating  fatally.  Most  animals  may  be  infected 
artificially. 

Leishman-Donovan  bodies  are  called  with  a  question  mark  trypanosomes 
by  Novy  who  states  that  they  resemble  rounded  forms  of  trypanosomes,  showing 
a  nucleus  and  a  micro-nucleus.  They  have  no  undulating  membrane.  They 
are  called  piroplasma  by  Laveran.  Rogers  regards  them  as  belonging  to  the 
herpetomonas  group  and  not  to  the  trypanosomes.  They  are  the  cause  of 
Kala-Azar,  a  cachexial  fever  of  India. 


PATHOGENIC    PROTOZOA.  411 

Walker*  has  published  a  full  account  of  his  work  on  the 
cultivation  of  parasitic  amebae. 

While  giving  due  credit  to  the  large  amount  of  valuable 
work  done  by  others  in  the  study  of  amebae,  Walker  has 
perfected  the  methods  of  study  to  such  an  extent  as  to  make 
this  branch  of  investigation  now  within  the  range  of  exact 
observation.  Walker's  original  article  is  recommended  to 
those  who  wish  to  persue  the  subject  fully;  but  the  following 
description  taken  from  his  paper  may  serve  as  a  guide,  and 
may  perhaps  enable  the  student  to  begin  investigations  into 
this  subject  which  is  increasing  in  importance  daily. 

Cultivation  of  Amebce. — The  medium  best  suited  for  the 
growth  of  amebae  is  that  introduced  by  Musgrave  and  Cleggt 
which  consists  of: 

Agar 20.00 

Sodium  cHorid 0.3-0.5 

Extract  of  beef 0.3-0.5 

Distilled  water 1,000 

This  medium  is  prepared  as  ordinary  culture-media  for  the 
cultivation  of  bacteria,  except  that  the  reaction  should  be  i 
per  cent,  alkaline  instead  of  the  1.5  usually  employed  for 
bacteria.  Ameba  cultivated  upon  this  medium  will  after- 
wards grow  less  readily  than  at  first  upon  liquid  media,  but 
they  grow  more  readily  upon  the  solid  medium  than  in  liquid 
media  when  transplanted  from  the  latter  to  the  former. 
Ameba  fecalis  is  the  only  one  which  grows  on  an  acid  medium. 
All  ameba  require  the  presence  of  bacteria  in  the  culture- 
medium  and  it  seems  that  the  bacteria  are  either  eaten  by  the 
ameba  or  that  they  transform  the  medium  in  some  way  so  as 
to  make  it  assimilable  by  the  amebae.  Moisture  is  even  more 
essential  for  the  growth  of  amebae  than  for  that  of  the  bacteria. 

*Journ.  Med.  Research.  Vol.  XVII.,  No.  4-  (New  Series,  Vol.  XII.  Feb., 
1908.  pp.  379-457.) 

fDept.  of  the  Interior.  Bureau  of  Gov.,  Labs.,  Biolog.  Lab.,  No.  18.  Oct., 
1904.  Manila  Bu.  Pub.  Printing. 


4I2  MANUAL    OF    BACTERIOLOGY. 

The  water  which  squeezes  out  of  the  agar  when  the  latter 
solidifies  is  sufficient  if  it  is  not  allowed  to  evaporate.  A 
glass  jar  provided  with  a  cover  and  containing  a  vessel 
holding  water  may  be  employed  for  slant  cultures.  For 
Petri  dish  cultures  a  battery  jar  inverted  in  a  shallow  dish 
of  water  will  serve  to  keep  the  cultures  moist.  Oxygen  is 
essential  to  the  growth  of  the  amebae.  They  grow  best  at  about 
20°  C.  to  25°  C.  but  they  will  also  grow  at  37.5°  C. 

ISOLATION    OF    PURE    CULTURES    OF    AMEBAE. 

The  amebae  grow  only  upon  the  surface  of  the  solid 
medium,  and  they  do  not  remain  fixed  as  do  the  bacteria  by 
the  setting  of  the  agar,  but  creep  about  from  place  to  place. 
It  is  therefore  necessary  to  resort  to  special  methods  to  secure 
cultures  consisting  of  one  kind  of  ameba  only.  If  an  agar 
Petri  plate  previously  poured  and  allowed  to  harden  is  stroked 
with  a  platinum  needle  in  several  parallel  lines  with  material 
containing  ameba  the  lines  made  last  will  show  scattered 
individual  ameba  cells.  The  needle  is  dipped  only  once  into 
the  material  to  be  studied.  The  plate  is  examined  with  the 
low  power  of  the  microscope,  and  a  single  ameba  separated 
from  all  the  rest  is  located.  A  high  power  lens  is  then  turned 
into  position  and  run  down  until  it  comes  in  contact  with  the 
ameba  selected.  In  this  way  the  ameba  is  picked  up  on  the 
end  of  the  lens  and  may  be  transplanted  to  fresh  media  to 
grow  out  and  form  a  culture  consisting  of  the  progeny  of  this 
one  cell.  Another  method  of  obtaining  a  pure  culture  of 
amebae  is  to  locate  a  cell  as  just  described  with  the  low  power 
of  the  microscope  but  with  the  Petri  dish  inverted.  The 
location  is  marked  around  on  the  bottom  of  the  inverted  dish 
with  a  wax  pencil  or  with  ink.  The  dish  is  now  turned  right 
side  up,  and  the  place  marked  out  as  the  location  of  the  cell 
is  inclosed  by  drawing  a  ring  of  vaseline  around  it.  This 
prevents  the  ameba  from  wandering  out,  and  also  prevents 


PATHOGENIC    PROTOZOA.  413 

other  species  from  wandering  in.  After  isolation  by  method 
just  described  the  cell  is  allowed  to  multiply  where  it  is,  and 
after  it  has  done  so,  as  shown  by  observation  with  the  micro- 
scope, further  culture  may  be  made  on  fresh  tubes  or  plates. 
These  cultures  are  pure  only  in  the  sense  that  they  contain 
only  one  kind  of  ameba,  but  they  require  the  presence  of 
bacteria  for  growth  as  has  been  elsewhere  stated. 

Study  of  the  Amebce  on  Cover-glasses. — Hill's  hanging 
block  method  is  not  well  suited  to  the  purpose  of  studying 
the  amebae  for  the  reason  that  these  grow  only  upon  the 
surface  but  agar  spred  in  a  thin  film  on  a  cover-glass  answers 
the  purpose  well.  The  method  of  procedure  is  to  spread  the 
melted  agar  on  the  cover-glass,  and  protect  it  against  contami- 
nation from  the  air  by  placing  it  under  a  flamed  watch  glass. 
The  spreading  is  affected  with  a  large  platinum  loop.  The 
agar  is  inoculated  with  the  ameba,  and  the  cover-glass  onto 
which  it  is  attached  is  placed  on  a  hollow  slide  in  the  manner 
used  in  making  the  usual  hanging  drop  cultures  of  bacteria. 
After  the  vaseline  is  streaked  around  the  hollow  in  the  slide, 
the  latter  may  be  pressed  down  over  the  cover-glass  containing 
the  inoculated  culture  medium,  or  the  cover-glass  may  be 
turned  over  on  to  the  slide  in  the  usual  way. 

Reproduction  in  the  Genus  Ameba. — Studied  in  the 
"hanging  plate"  method  just  described,  ameba  are  seen  to 
pass  through  several  stages.  The  ameboid  stage  may  give 
rise  to  other  ameboid  forms  by  division,  or  to  a  resting  stage 
through  encystment,  or  finally  to  the  production  of  young 
amebae  through  spore  formation. 

In  the  process  of  development  first  mentioned  the  cell  comes 
to  rest,  becomes  rounded  and  then  oval,  and  finally  becomes 
constricted  into  a  mother  and  a  daughter  cell  by  transverse 
division.  Sometimes  the  two  cells  remain  attached  by  a 
thin  thread  of  protoplasm  which  stretches  to  several  times  the 
diameter  of  the  cells.  The  cells  are  sometimes  pulled  together 


414  MANUAL    OF    BACTERIOLOGY. 

by  the  retraction  of  this  thread  which  thickens  as  it  shortens. 
The  cells  may  pull  apart  and  be  drawn  together  again  by  this 
thread  of  protoplasm  several  times  in  the  course  of  an  hour. 
Division  of  the  nucleus  is  in  some  cases  typically  amniotic. 
By  dropping  cover-glasses  in  fixing  fluid  at  various  stages  of 
the  process  certain  stages  can  be  studied.  The  encystment 
mentioned  above  takes  place  sooner  or  later  in  all  culture  after 
longer  or  sorter  period  of  multiplication  and  independently 
of  the  amount  of  moisture,  but  drying  out  hastens  the  proc- 
ess. Encysted  amebae  transplanted  to  fresh  culture-medium 
usually  begins  to  show  signs  of  awakening  activity  in  twenty- 
four  hours.  These  consist  of  agitation  of  the  granules  of  the 
protoplasm,  and  finally  pulsation  of  the  vacuole.  If  too  much 
of  the  old  bacterial  growth  is  transplanted  on  to  the  fresh 
culture  medium,  the  cysts  may  fail  to  germinate.  No  multi- 
plication takes  place  within  the  cyst.  The  ameba  leaves  the 
cyst  behind  as  an  empty  shell. 

Spore  Formation. — Spore  formation  in  amebae  takes  place 
usually  within  48-72  hours  after  they  are  transplanted  to 
fresh  medium;  but  it  varies  as  to  time  in  different  species. 
They  first  appear  as  fine,  brightly  refractive  granules,  which 
become  larger  and  larger,  by  the  aggregation  of  chromidia 
or  by  becoming  surrounded  by  a  layer  of  cytoplasm  until  they 
may  become  as  large  as  the  nucleus,  and  in  fact  have  been 
mistaken  by  some  observers  for  multiple  nuclei.  The  spores 
vary  in  size  from  .7  to  2  mikra,  and  they  stain  faintly  and 
are  more  readily  decolorized  than  the  amebae.  They  are 
extruded  from  the  ameba  in  24-48  hours  from  the  time  of 
tHeir  first  appearance,  but  an  ameba  may  become  encysted 
before  the  spores  are  extruded  which  has  probably  led  to 
the  error  of  supposing  that  the  encysted  ameba  may  form 
spores.  The  development  of  the  spore  into  the  ameba  is 
direct;  the  thickening  of  the  wall  described  by  some  does  not 
take  place  if  the  conditions  of  growth  are  favorable.  The 


PATHOGENIC    PROTOZOA.  415 

spore  grows  larger  and  larger,  and  finally  the  nucleus  and  the 
vacuole  make  their  appearance.  The  spores  probably  contain 
cromatin  granules.  No  sexual  process  was  observed. 

Methods  of  Making  Permanent  Preparations. — Prepara- 
tions may  be  obtained  by  applying  a  perfectly  cleaned  cover- 
glass  to  the  Petri  dish  culture,  and  dropping  it  at  once 
into  fixing  fluid,  but  a  better  way  is  to  drop  a  hanging  plate 
culture  into  the  fixing  fluid.  The  hanging  plate  must  in  this 
case  have  been  prepared  on  a  very  clean  cover-glass,  or  it 
must  have  been  made  on  cover-glasses  which  have  been 
covered  with  a  very  thin  layer  of  egg  albumen  before  the  agar 
is  spread  on  it,  otherwise  the  film  is  apt  to  become  detached. 
The  stain  recommended  specially  by  Walker  is  Mallory's 
chloride  of  iron  hematoxylin.* 

From  various  sources  Walker  obtained  44  different  cultures 
which  he  separates  into  10  distinct  species  using  as  criteria 
the  characters  of  the  ameboid,  the  encysted,  and  the  sporu- 
lating  stages.  A.  coli  Loesch  (man) ;  A.  hominis  sp.  n.  (man) ; 
A.  copayae  sp.  n.  (guinea-pig) ;  A.  musculi  sp.  n.  (house  mouse) ; 
A.  gallopavonis  sp.  n.  (turkey);  A.  ranas  sp.  n.  (frog);  A. 
muris  Grassi  (mouse  and  rat);  A.-intestinalis  sp.  n.  (various 
animals) ;  A.  enterica  sp.  n.  (various  animals) ;  A.  fecalis  sp.  n. 
(wide  range  of  hosts). 

Ameba  coli  Loesch  has  a  number  of  synonyms.  It  is  the  organism  described 
by  Councilman  and  Lafleur  as  A.  dysenteriae  and  taken  by  them  and  many 
others  to  be  the  cause  of  amebic  dysentery. 

Ameboid  stage,  circular  in  outline  when  at  rest;  oblong,  ligulate,  or  irregular 
when  in  motion;  size  9-20  mikrons  when  in  resting  stage;  pseudopodium 
normally  single,  lobose;  ectoplasm,  hyaline,  apparently  only  in  the  pseudo- 
podium;  entoplasm  finely  granular;  nucleus  circular  or  oval,  plastic,  3-5  mikrons, 
surrounded  by  a  narrow  halo;  vacuoles  one  to  several,  non -contractile.  Encysted 
stage  appearing  late  and  slowly  in  cultures;  cysts  circular,  6-9  mikrons;  wall 
single,  double  contoured,  regular;  contents  finely  granular;  nucleus  not  visible. 
Sporulation  not  frequent;  spores  never  numerous,  spheroidal  .7-2  mikrons. 
Movement  rather  active.  Habitat,  intestinal  tract  of  man. 

Ameba  cobaya  Walker.  Ameboid  stage  circular  when  at  rest,  oval,  oblong, 
ligulate,  less  frequently  irregular,  when  in  motion;  15.5-25  mikrons  in  the  resting 
condition;  pseudopodium  normally  single,  lobose;  ectoplasm,  hyaline  extensive; 
entoplasm  coarsely  granular;  nucleus  circular  but  plastic,  surrounded  by  a 

*  Mallory  and  Wright.     Pathological  Technic.     1904. 


416  MANUAL    OF    BACTERIOLOGY. 

hyaline  halo,  otherwise  homogeneous;  a  single  contractile  vacuole.  Encyst- 
ment  early  in  cultures;  cysts  circular  or  oval;  7.5-14  mikrons;  wall  single, 
double  contoured  regular;  contents  coarsely  granular;  nucleus  not  visible. 
Sporulation  frequent;  spores  numerous  spheroidal,  .7-2  mikrons.  Movement 
very  active.  Habitat,  intestinal  tract  of  the  guinea-pig. 

Ameba  hominls  Walker.  Ameboid  stage  circular  when  at  rest,  oval,  obovate 
or  oblong  when  in  motion,  6.5-15.4  mikrons  at  rest;  pseudopodium  single  lobose; 
ectoplasm  apparently  only  in  the  pseudopodium,  hyaline;  entoplasm  finely 
granular;  nucleus  circular,  surrounded  by  a  narrow  halo  2.8-4.3  mikrons; 
a  single  contractile  vacuole.  Encysted  stage  assumed  late  and  slowly  in  cultures; 
cyst  circular,  4.6-7.7  mikrons  wall  single,  double  contoured,  regular;  contents 
finely  granular,  nucleus  not  visible.  Sporulation  very  frequent;  spores  spher- 
oidal, numerous,  .3-. 8  mikrons.  Habitat,  intestinal  tract  of  man. 

Ameba  musculi  Walker.  Ameboid  stage  circular  in  resting  stage;  oval, 
oblong,  obovate,  or  slightly  irregular  when  in  motion;  7.7-13.9  mikrons  when 
at  rest;  pseudopodium,  single,  lobose;  ectoplasm  occupying  about  half  the 
ameba,  hyaline;  entoplasm  containing  a  few  coarse  granules;  nucleus  spheroidal 
but  plastic,  surrounded  by  a  broad  halo,  otherwise  homogeneous,  2.3-3.1 
mikrons;  single  contractile  vacuole.  Encysted  stage  assumed  only  after 
a  very  long  time  and  very  slowly  in  cultures;  cysts  circular  or  oval,  6.2-7.7 
mikrons;  wall  single,  double  contoured,  regular;  contents  granular,  nucleus  not 
visible.  Sporulation  rather  infrequent;  spores  not  very  numerous,  spheroidal, 
1-2.3  mikrons.  Movement  fairly  active,  amebas  wandering  far  beyond  bacterial 
growth.  Habitat,  intestinal  tract  of  house  mouse. 

Ameba  gallopavonis  Walker.  Ameboid  stage  circular  when  at  rest,  oval 
oblong  or  irregular  when  in  motion,  18.5-30.8  mikrons  in  resting  stage;  pseudo- 
podium normally  single,  short  blunt;  ectoplasm  more  or  less  distinct,  hyaline; 
entoplasm  granular;  nucleus  circular,  3.4-5.1  mikrons,  surrounded  by  a  medium 
wide  halo;  a  single  contractile  vacuole;  encysted  stage  assumed  rather  slowly 
in  cultures;  walls  two;  inner  wall  very  irregular,  as  if  the  ameba  had  become 
encysted  in  the  ameboid  shape;  outer  wall  fairly  regular,  separated  from  the 
inner  wall  but  touching  it  at  the  angles,  12.3-20.8  mikrons.  Sporulation 
occasional;  spores  few,  5-9  spheroidal,  1.2-1.5  mikrons  in  diameter.  Move- 
ment sluggish.  Habitat,  intestinal  tract  of  the  turkey. 

Ameba  ranee  Walker.  Ameboid  stage  circular  when  at  rest,  oval,  oblong 
or  irregular  when  in  motion,  10.8-15.4  mikrons  when  at  rest;  pseudopodium 
single,  broad;  ectoplasm  slightly  evident  in  the  resting  ameba,  hyaline;  ento- 
plasm containing  scattered  coarse  granules;  nucleus  circular,  1.5-3  mikrons 
with  a  relatively  broad  halo,  otherwise  homogeneous;  a  single  contractile 
vacuole.  Encysted  stage  assumed  slowly  in  cultures;  cysts  circular  in  outline, 
9.8-14  mikrons;  wall  single,  double  contoured,  regular;  contents  granular; 
Sporulation  moderately  frequently;  spores  8-16,  spheroidal,  1.2-1.5  mikrons. 
Movement  sluggish.  Ameba  do  not  wander  from  bacterial  growth  in  culture. 
Habitat,  intestinal  tract  of  frog. 

Ameba  muris  Grassi.  Ameboid  stage  circular  when  at  rest,  5.4-8.5  mikrons, 
oval,  oblong,  occasionally  slightly  irregular  when  in  motion;  pseudopodium, 
single  lobose;  ectoplasm  and  entoplasm  scarcely  differentiated;  hyaline  with 
fine,  scattered  granules;  nucleus  very  small,  spheroidal,  with  a  narrow  halo. 
Encysted  stage  late,  cysts  circular,  3.9-5.4  mikrons;  wall  single,  double,  con- 
toured, regular,  concentrically  separated  from  the  contents  in  old  cysts;  contents 
finely  granular.  Sporulation  rather  late;  spores  minute,  spheroidal.  Move- 
ment sluggish;  amebae  do  not  wander  from  bacterial  growth.  Habitat,  intestinal 
tract  of  mouse. 

Ameba  intestinalis  Walker.  Ameboid  stage  circular  when  at  rest;  oval, 
oblong,  or  less  often  irregular  when  in  motion;  size  9-26  mikrons  when  at  rest; 


PATHOGENIC    PROTOZOA.  417 

pseudopodium  single,  short,  broad;  ectoplasm  scarcely  apparent  except  in 
pseudopodia,  hyaline,  entoplasm  coarsely  granular,  frequently  vacuolated; 
nucleus  circular,  but  somewhat  plastic,  surrounded  by  a  broad  halo,  otherwise 
homogeneous,  3.1-6.2  mikrons  in  diameter;  a  single  contractile  vacuole.  En- 
cysted stage  assumed  early,  and  rapidly;  cysts  irregular  from  the  beginning 
walls  two;  inner  wall  irregularly  polygonal,  scalloped  or  ovoid;  outer  wall 
wrinkled  and  touching  in  the  inner  wall  at  its  angles;  contents  of  cysts  coarsely 
granular,  nucleus  frequently  visible.  Sporulation  moderately  frequent;  spores 
numerous,  spheroidal,  .5-1.5  mikrons.  Movement  not  very  active,  but  the 
amebas  wander  widely  over  the  culture  medium.  Habitat,  intestinal  tract  of  the 
horse,  pig,  cat,  turkey  and  perhaps  other  animals. 

Ameba  enterica  Walker.  Ameboid  stage  circular  when  at  rest,  oblong  or 
irregular  when  in  motion;  9.2-15.4  mikrons  in  diameter  when  at  rest;  pseudo- 
podium normally  single,  blunt,  narrow;  ectoplasm  apparent  only  in  the 
pseudopodia,  hyaline;  entoplasm  finely  granular;  nucleus  circular  in  outline, 
3.1-6.2  mikrons,  surrounded  by  a  broad  halo,  otherwise  homogeneous;  a  single 
contractile  vacuole.  Encysted  stage  assumed  late  and  slowly;  cysts  at  first 
circular,  later  slightly  irregular;  walls  two;  inner  wall  circular,  double  contoured, 
at  first  circular,  later  becoming  polygonal,  or  lobed  in  outline;  contents  finely 
granular,  nucleus  not  visible;  sporulation  rather  frequent;  spores  numerous, 
spheroidal,  .8-2  mikrons.  Motility  moderately  active.  Habitat,  intestinal  tract 
of  the  rabbit,  cat,  rat,  mouse,  turkey,  and  perhaps  other  animals. 

Ameba  fecal. s  Walker.  Ameboid  stage  circular  when  at  rest,  oval,  oblong 
or  irregular  when  in  motion,  6.2-15.4  mikrons;  pseudopodium  normally  single, 
lobose;  ectoplasm  and  entoplasm  not  differentiated,  finely  granular,  frequently 
vacuolated,  feebly  refractive;  nucleus  small  spheroidal,  surrounded  by  a  narrow 
halo,  a  single  contractile  vacuole.  Encysted  stage  assumed  rather  early  in 
cultures;  cysts  circular  or  slightly  irregular,  3.1-7.8  mikrons;  wall  single,  im- 
pervious to  stain;  contents  regularly  contract  from  one  or  two  segments  of  the 
wall.  Sporulation  rather  infrequent;  spores  minute.  Movement  slow.  Habi- 
tat, intestinal  tract  of  various  vertebrate  animals,  and  probably  also  free. 

Other  amebae  have  been  described  in  connection  with  various  diseases  of 
animals,  but  owing  to  lack  of  exhaustive  descriptions  these  could  not  be  brought 
under  the  classifications  of  Walker  given  above,  though  doubtless  some  of  them 
in  reality  belong' to  one  or  the  other  of  the  species  given  above.  Ameba  dysen- 
teries of  Councilman  and  Lafleur  dysenterica,  Kartulis,  coli  felis  Quinke  and  Roos, 
coli  mills  ebenda,  intestini  vulgaris  ebenda,  lobosa  coli  Celli  e  Fiocca,  entameba 
hominis  Casagrandi  e  Babagallo,  coli  Schaudinn  are  all  regarded  as  identical 
with  Ameba  coli  Loesch.  Unidentified  or  identified  with  reserve  are  ameba 
bovis  Liebetanz,  bucallis  Sternberg,  bentali  Grassi,  gingivalis  Gross,  ka^tulisi 
Doflein,  miurai  Ijima,  pulmonalis  Arlault,  ranarum  Grassi,  urogenitalis  Baelz, 
buccalis  Prowazek,  undulans  Csatellanil,  gemmipara  Schaudinn. 

The  Malarial  Parasite*  (Plasmodium  or  Haematozoon 
Malariae). — The  organisms  of  malaria  consist  of  at  least  three 
different  species,  each  associated  with  one  of  the  three  types 
of  malarial  fever:  The  tertian  parasite  with  benign  tertian 
malarial  fever,  the  parasite  reaching  maturity  in  forty-eight 

*See   Thayer  and  Hewetson.     The  Malarial  Fevers  of   Baltimore.     Johns 
Hopkins    Hospital    Reports.     Vol.     V.     1895.     Thayer.     Lectures     on     the 
Malarial  Fevers.     New  York.     1897. 
27 


4i8 


MANUAL    OF    BACTERIOLOGY. 


hours;  the  quartan  parasite  with  benign  quartan  malarial 
fever,  the  cycle  of  development  requiring  seventy- two  hours; 
and  the  estiva-autumnal  parasite  with  malignant,  estivo- 
autumnal  fever,  developing  to  maturity  in  a  variable  period  of 
from  twenty-four  to  forty-eight  hours.  The  parasites  are 


FIG.  1 06. 


FIG.  107. 


FIG.  108. 


FIG.  109 


FIGS.  106-109. — Malarial  parasites  in  various  stages.     (X  1000.) 
Figs.  106,  107  and  108  are  tertian  parasites.     Fig.  108  shows  the  completion 
of  segmentation.     Fig.  109  is  the  cresccntic  form  of  the  estivo-autumnal  parasite 


studied  to  best  advantage  in  a  drop  of  fresh,  fluid  blood  placed 
between  a  cover-glass  and  slide  and  examined  with  an  oil- 
immersion  objective.  For  method  of  making  and  staining 
dry  preparations  see  pages  45  and  108. 

Tertian  Parasite. — This  appears  in  its  youngest  form  as  a 


PATHOGENIC    PROTOZOA. 


419 


small,  round,  colorless,  hyaline  body  within  the  red  corpuscle, 
seen  during  and  just  after  the  chill  of  the  disease.  This  body 
may  be  actively  ameboid,  suddenly  changing  its  contour  into 
various  forms.  Its  size  gradually  increases,  and  fine,  dark, 
actively  motile,  dancing  pigment  granules  begin  to  appear  at 
its  periphery. 

The  red  corpuscle  harboring  the  parasite,  with  the  growth 
of  the  latter,  becomes  gradually  paler  and  expands  in  size. 
The  parasite  as  it  grows  loses  its  earlier  ameboid  movement, 
and  the  pigment  granules,  still  actively  motile,  accumulate. 
Near  the  end  of  forty-eight  hours  the  organism  finally  fills 
the  red  corpuscle,  only  a  faint  rim  indicating  the  latter.  The 
ripe  parasite  now  divides  it  into  from  fifteen  to  twenty-five 
small,  round,  hyaline  spores,  which  are  arranged  somewhat 
radially  about  the  pigment  granules  which*  have  lost  their 
motility  and  become  concentrated  in  a  clump  at  the  center  of 
the  spore-forming  organism.  The  spores  finally  break  apart 
and  scatter,  each  destined  to  invade  a  red  corpuscle  and 
start  anew  the  cycle  of  development.  This  cycle  may  be 
repeated  over  and  over  again,  producing  a  corresponding 
number  of  malarial  paroxysms. 

Certain  full-grown  parasites  do  not  complete  the  cycle  of  development  by 
sporulation,  as  described,  but,  breaking  loose  from  the  corpuscle,  remain  as 
"extracellular"  bodies.  These  are  seen  chiefly  after  the  paroxysm  as  large, 
round,  pale  bodies  containing  numerous  dancing  pigment  granules  scattered 
through  their  substance.  They  ultimately  degenerate  and  disappear.  Some 
of  these  extracellular  forms  may  be  seen  to  develop  long  slender  processes, 
flagella,  having  a  very  active  whip-like  motion.  Flagella  are  never  observed 
in  perfectly  fresh  blood,  but  develop  only  after  the  blood  has  been  drawn  some 
time,  usually  fifteen  or  twenty  minutes. 

The  extracellular  forms  of  the  parasite,  the  gametes,  incapable  of  further 
development  in  their  human  intermediate  host,  can  continue  their  life  cycle 
only  when,  by  chance,  they  happen  to  be  sucked  into  the  body  of  a  mosquito 
of  the  genus  Anopheles,  the  definite  host,  in  which  they  undergo  a  second  com- 
plete sexual  cycle  of  development  with  the  ultimate  production  of  spores  or 
sporozoi'ds.  When  in  turn  the  spores  chance  to  be  inoculated  into  the  blood 
of  man  by  the  bite  of  an  infected  Anopheles,  the  man  becomes  infected,  and 
the  cycle  of  development  in  the  red  corpuscle,  already  outlined,  commences, 
The  second  or  sexual  cycle  of  the  parasite  in  the  mosquito,  here  described 
for  the  tertian  organism,  applies  as  well  to  the  other  varieties  of  the  malarial 
organism,  namely  the  quartan  and  the  estivo-autumnal  forms,  in  the  case  of 


420  MANUAL    OF    BACTERIOLOGY. 

each  starting  from  the  extracellular  mature  forms  of  the  organism  found  in  the 
blood  of  the  human  host.* 

Quartan  Parasite. — This  resembles  quite  closely  the  ter- 
tian parasite,  but  differs  from  it  in  certain  respects.  The 
young,  hyaline,  intracorpuscular  parasite  is  more  highly 
refractive,  its  ameboid  motion  is  less  marked  and  more  slug- 
gish, and  the  pigment  granules  are  darker,  much  coarser, 
and  have  very  slight  motility.  The  infected  red  corpuscles 
are  usually  somewhat  contracted  instead  of  swollen,  and  their 
color  is  apt  to  be  darker,  assuming  a  bronzed  hue.  The 
full-grown  parasite  is  much  smaller  than  the  corresponding 
form  of  the  tertian,  approximating  the  size  of  a  normal  red 
corpuscle.  As  segmentation  begins,  a  characteristic  appear- 
ance develops  which  distinguishes  the  quartan  organism, 
namely,  the  coarse  pigment  granules  are  drawn  toward  the 
center  of  the  parasite  in  certain  converging  straight  paths, 
giving  a  stellate  arrangement  to  the  pigment,  until  finally  it 
becomes  clumped  entirely  at  the  center  in  a  solid  mass.  The 
segmenting  forms  of  the  quartan  parasite  thus  present  a  more 
symmetrical  arrangement  of  the  spores,  which  often  resemble 
the  petals  of  a  "  marguerite."  These  spores  are  oval  and  num- 
ber only  from  six  to  twelve,  being  fewer  than  those  of  the  ter- 
tian segmenting  parasite.  The  quartan  extracellular  forms 
are  smaller  than  those  of  the  tertian,  being  about  the  size  of  a 
red  corpuscle,  and  contain  coarse  pigment  granules  in  active 
motility  until  degeneration  occurs.  Flagella"  may  develop  from 
certain  extracellular  forms.  The  entire  development  of  the 
quartan  parasite  occupies  about  seventy-two  hours. 

Estivo-autumnal  Parasite. — This  parasite  develops  to  ma- 
turity in  from  twenty-four  to  forty-eight  hours,  and  is  usually 
regarded  as  representing  a  single  species,  though  certain 
observers  claim  to  distinguish  two  distinct  varieties.  The  usual 
description  of  a  single  variety  is  here  adopted.  The  youngest 

*Lyon.  The  Inoculation  of  Malaria  by  the  Mosquito.  A  Review  of  the 
Literature.  Medical  Record.  February  17,  1900. 


PATHOGENIC    PROTOZOA.  421 

forms  (hyaline  bodies)  resemble  those  of  the  tertian  and  quar- 
tan organisms,  but  are  distinctly  smaller  and  more  highly 
refractive.  They  often  present  a  ring-like  appearance.  They 
are  ameboid.  Pigment  granules  later  appear  at  their  per- 
iphery, but  are  exceedingly  minute  and  scanty,  seldom  more 
than  one  or  two  being  seen.  These  granules  have  little  or  no 
motility,  and  in  fact  are  with  difficulty  made  out.  The  hyaline 
bodies  remain  small,  seldom  exceeding  one-third  the  diameter 
of  a  red  corpuscle.  The  infected  corpuscle  is  apt  to  be  crenated, 
shrunken  and  dark.  These  are  the  forms  seen  in  the  circu- 
lating blood  in  early  infections;  the  mature  forms,  with  the 
exception  of  the  extracellular  forms,  developing  in  the  spleen 
and  bone- marrow,  rarely  reach  the  general  circulation.  Blood 
from  the  spleen  shows  the  full-grown  forms  in  abundance. 
The  segmenting  forms  resemble  those  of  the  tertian  parasite 
both  in  the  numbers  of  the  segments  and  in  their  arrangement 
but  are  much  smaller  in  the  aggregate,  as  well  as  in  the  indi- 
vidual segments. 

After  the  fever  has  lasted  about  one  week,  extracellular 
forms  make  their  appearance  in  the  circulating  blood.  These 
are  crescentic,  ovoid  or  small  round  bodies,  containing  coarse 
pigment  granules  at  their  center,  generally  arranged  in  a 
ring.  The  crescents  and  ovoid  bodies  are  highly  refractive 
and  are  in  length  about  equal  to  the  diameter  of  a  red  cor- 
puscle, sometimes  larger.  The  round  forms  are  smaller  than 
a  red  corpuscle,  with  the  pigment  arranged  centrally  in  a 
ring.  They  may  become  flagellated  after  the  blood  has 
remained  outside  the  body  for  some  minutes.  Any  of  the 
extracellular  bodies  may  show  remnants  of  the  red  corpuscle 
attached  to  its  side,  like  a  bib.  The  extracellular  forms  are 
concerned  in  the  cycle  of  development  of  the  organism  in  the 
mosquito,  and  are  sterile  in  the  human  body.  They  are 
exceedingly  resistant  to  quinine  and  may  continue  in  the  blood 
for  long  periods  of  time. 


422  MANUAL    OF    BACTERIOLOGY. 

Melaniferous  leukocytes  are  seen  in  the  blood,  being  espe- 
cially abundant  after  the  paroxysm  in  all  forms  of  malarial 
infection.*  These  are  phagocytes  which  have  taken  up  the 
pigment  granules  liberated  by  the  disintegration  of  the 
erythrocytes. 

Small-pox  and  Vaccinia. — Micrococci  of  various  sort 
have  been  found  in  the  pustules  of  small-pox  and  vaccinia, 


* 

"         * 

FIG.  no. — Trypanosomes  in  the  blood  of  the  rat.     Romanowsky  stain. 
(X  1000.) 

but  indicate  only  a  secondary  infection.  Other  microorgan- 
isms have  been  described.  The  most  important  are  certain 
bodies  often  considered  protozoa.  In  both  small-box  and 
vaccinia  small,  round  homogeneous  bodies,  2  to  4  /*  in  diam- 
eter, have  been  found  in  the  epithelial  cells  of  the  vesicles. 
Inoculation  of  vaccine  lymph  into  the  rabbit's  cornea  leads  to 
the  production  of  similar  bodies  in  the  epithelial  cells  of  the 

*See  also  Ewing.     Journal  Experimental  Medicine.     Vols.  V.  and  VI. 


PATHOGENIC    PROTOZOA.  423 

cornea.  W.  Reed*  found  small  ameboid  bodies  in  the  blood 
in  cases  of  small-pox  and  vaccinia.  Vaccine  virus  that  has 
been  filtered  through  the  Chamberland  or  Berkefeld  filter  is 
still  active,  but  somewhat  diminished  in  power,  t  From  this 
it  may  be  presumed  that  the  organism  causing  it  is  not  too 
small  to  be  seen  with  the  microscope. 

Councilman,  Magrath  and  Brinkerhoff,J  as  a  result  of 
recent  studies,  believe  that  the  bodies  above  mentioned  are 
protozoa.  Segmentation  of  the  bodies  is  described,  resulting 
in  the  formation  of  spore-like  bodies.  The  spore-like  bodies 
undergo  a  further  or  second  cycle  of  development  within  the 
nucleus.  The  second  cycle  also  ends  in  segmentation.  The 
two  cycles  were  seen  in  small-pox;  in  vaccinia,  only  the  first 
or  extranuclear  bodies  were  observed. 

Guarnieri§  was  the  first  to  describe  these  bodies  with 
accuracy,  and  give  to  them  the  name  cytoryctes  variola.  He 
found  them  by  staining  with  carmine,  hematoxylin,  and  saf- 
ranin  in  the  deeper  layers  of  the  epithelium  in  cases  of  vaccinia 
and  small-pox.  Much  opposition  to  Guarnieri's  inter- 
pretation of  this  observation  was  aroused,  but  on  the  other 
hand,  in  the  epicrisis  to  a  series  of  articles  on  small-pox  and 
vaccinia  by  himself,  Magrath,  Brinkerhoff,  Tyzzer,  Southard, 
Thompson,  Bancroft,  and  Calkins. ||  Councilman  strongly 
supports  the  view  that  the  Guarnieri  bodies  are  living  organ- 
isms and  the  cause  of  the  diseases.  He  believes  both  small- 
pox and  vaccinia  to  be  caused  by  the  same  parasite,  and  that 
while  certain  form  of  cell  degeneration  may  closely  simulate 
Guarnieri  bodies  that  they  are  not  identical  with  these. 

^Journal  Experimental  Medicine.  Vol.  II.  p.  515.  See  also  Anna  Williams 
and  Flournoy,  and  W.  H.  Park.  New  York  University  Bulletin  Medical 
Sciences.  Vol.  II.  October,  1902. 

fNegri.  Zeitschr.  f.  Hygiene.  Vol.  LIV.,  No.  3.  Oct.  26,  1906.  p.  32?- 
Also  see  Bull,  de  I'Inst.  Past.  Vol.  V.,  No.  i.  Jan.  15,  1907-.  P- 23- 

t  Journal  Medical  Research.     Vol.  IX.     May,  1903-     Jtnd.  Vol.  *A- 

\Arch.  per  le  Sci.  Med.  XXVI.  p.  403;  CentralUt.  f.  Bakteno.  XVI.  p. 
299. 

\\Journ.  Med.  Research.     Vol.  II.,  No.  i.     pp.  7~361- 


424  MANUAL    OF    BACTERIOLOGY. 

Ewing*  on  the  contrary  comes  to  the  conclusion  from  his 
study  of  the  subject,  in  which  he  corroborates  and  extends  the 
observations  of  others,  that  the  bodies  are  probably  degenerated 
tissue  cells.  The  fact  that  the  forms  met  with  are  peculiar  to 
variola  and  to  vaccinia,  not  found  in  other  morbid  processes 
nor  in  healthy  tissue,  Ewing  finds  is  no  convincing  proof  of  the 
organized  character  of  the  bodies;  for  the  forms  of  cell  degener- 
ation found  in  diphtheria,  in  measles,  in  glanders,  in  rabies, 
are  all  peculiar  and  characteristic  in  these  diseases.  No 
other  agent  has  been  found  which  will  cause  the  same  form 
of  cell  degeneration  which  is  caused  by  the  toxin  of  diphtheria, 
and  this  is  true  in  regard  to  other  diseases  than  diphtheria. 

It  is  thus  evident  that  competent  authorities  are  not  as  yet 
agreed  upon  the  nature  of  the  organism  causing  small-pox. 
Still  it  seems  settled  that  there  are  characteristic  bodies  which 
are  always  found  in  the  disease  whether  these  be  living  para- 
sites or  merely  peculiar  forms  of  cell  degeneration.  These 
bodies  are  also  found  in  vaccinia,  and  in  the  lesions  produced 
by 'the  inoculation  of  monkeys,  and  also  in  the  lesions  of  the 
cornea  of  rabbits  which  have  inoculated  in  the  eye  with  small- 
pox or  vaccine  virus. 

SCARLET  FEVER  AND  MEASLES. 

Fieldf  obtained  in  specimens  from  the  skin  taken  at  autopsy  and  in  the 
serum  secured  intravitam  by  the  use  of  a  small  blister-plaster  in  cases  of  scarlet 
fever  and  measles  preparations  showing  protozoon-like  bodies  previously 
described  by  MalloryJ  in  material  secured  at  autopsy  in  cases  of  scarlet  fever 
only.  Field  used  many  different  kinds  of  stains,  and  found  Giemsa  to  give  the 
best  results ;  Hastings  almost  as  good.  The  bodies  which  are  of  interest  resemble 
closely  the  extracellular  forms  of  the  malarial  parasite.  They  show  a  pale  blue 
protoplasm  with  one  or  more  granules  which  vary  in  size  from  a  mere  point  to  a 
particle  occupying  almost  the  total  diameter  of  the  body.  In  four  cases  they 
were  arranged  so  as  to  imitate  the  malarial  rosettes.  Field  states  in  regard  to 
the  character  of  these  bodies,  that  while  it  cannot  be  stated  that  they  are  not 
protozoa  the  majority  of  thsm  arise  fiom  degenerating  cells.  The  bodies  in  the 
blister-fluid  resemble  other  granular  bodies  seen  in  the  blood  under  certain  con- 


*Ewing.     Journ.  Med.  Research.     Vol.    XII.  .  1904.     p.    509.     Vol.    XIII. 
1905.     pp.  233-252. 

jJourn.  Exper.  Med.     Vol.  VII.     Feb.-Nov.  1905.     pp.  343-350. 
t Journ.  Med.  Research..    Vol.  X.     1904.     p.  483. 


PATHOGENIC    PROTOZOA.  425 

ditions,  and  al  o  in  vaccine  lymph,  and  in  emulsions  from  tissues  and  in  exudates. 
He  therefore  regards  them  as  for  the  most  part,  if  not  wholly,  products  of  degen- 
erating tissue  cells  and  of  leukocytes. 


YELLOW  FEVER. 


It  has  already  been  indicated  (page  149)  that  the  study  of  cases  of  yellow 
fever  has  failed  to  prove  that  this  disease  is  caused  by  bacteria.  On  the  other 
hand,  evidence  that  it  is  transmitted  by  the  mosquito,  Stegomyia,  has  been 
increasing. 

Piroplasma  Bigeminum,  Pirosoma  Bigeminum,  Apio- 
soma  Bigeminum. — Theobald  Smith  was  the  first  to  describe 
this  parasite  which  is  found  in  the  blood  of  cattle  suffering  with 
cattle  fever,  though  Babes  reported  having  found  certain  cell 
inclusions  in  the  blood  of  sick  cattle  in  Roumania,  and  Smith 
and  Kilborn  made  extensive  studies  of  the  organism.  It  is 
pear-shaped  occurring  in  pairs  with  the  pointed  end  toward 
each  other,  each  half  of  the  pair  is  2  to  4  mikrons  long,  by  i^- 
to  2  mikrons  wide.  It  occurs  inside  the  red  corpuscles. 
Pseudopodia  are  described  by  Nuttall  and  Graham-Smith.* 
It  stains  well  with  the  basic  dyes  and  is  well  brought  out  by 
Romanowsky.  It  has  never  yet  been  cultivated.  It  is  spread 
from  sick  to  diseased  animal  by  a  tick,  Boophilus  bovis  sive 
annulatus,  in  America,  by  several  species  of  ticks  apparently 
in  Germany.  The  ticks  seem  to  serve  merely  as  carriers, 
the  organism  appears  to  undergo  no  development  in  the  body 
of  the  tick.  Other  animals  than  cattle  also  suffer  from 
infection  with  organisms  of  similar  description.  Sheep,  dogs, 
horses,  and  apes  have  been  found  to  harbor  a  parasite  of 
this  character.  It  has  been  stated  that  men  have  also  been 
found  with  a  similar  organism  in  the  blood  in  cases  of  "  Spotted 
fever"  in  Montana  as  reported  by  Wilson  and  Chowning.t 
But  StilesJ  failed  to  corroborate  this  statement.  Ricketts§ 
found  that  the  disease  is  transmitted  by  a  tick,  Dermacentor 

*Journ.  Hygiene.     Vol.  VI.     1906.     p.  586. 

tRep.  Montana  State  Board  of  Health.     1901-2.     Also  Journ.  Inject.  Dis- 
eases.    Vol.  I.,  No.  I.     Jan.  i,  1904.     pp.  31-57- 
J  Pub.  Health  and  Mar.  Hosp.  Bull.     No.  20,  1905. 
\Journ.  Inject.  Dis.     Vol.  IV  ,  No.  i.     Jan.  i,  1907.     pp.  I4I-I53- 


4^6  MANUAL    OF    BACTERIOLOGY. 

occidentalis,  both  male  and  female.  He  also  found  that  one 
attack  affords  a  high  degree  of  immunity.  The  virus  is 
not  filterable  through  a  Berkefeld  filter.  The  parasite, 
whatever  it  may  be,  is  found  in  the  body  fluids  generally. 
The  infectiousness  of  the  blood  is  largely  destroyed  by  grinding 
the  infectious  blood  in  a  ball-mill. 

Certain  peculiar  bodies  suggestive  of  parasites  have  been 
found  in  the  lesions  of  a  tropical  disease  known  as  "Delhi 
Boil.''  Wright*  gives  in  substance  the  following  description 
of  the  disease  and  the  bodies  alluded  to.  The  disease  resem- 
bles some  of  the  manifestations  of  syphilis  and  tuberculosis, 
and  is  held  to  be  infectious.  It  consists  of  multiple  nodules 
in  the  skin  which  finally  ulcerate.  It  lasts  for  months  or  for  a 
year  or  longer.  In  smear  preparations  made  from  material 
obtained  from  one  of  the  lesions,  fixed  in  methyl  alcohol,  and 
stained  by  Wright's  modification  of  the  Romanowsky  a  large 
number  of  the  bodies  first  described  by  Cunningham  and 
believed  by  him  to  be  living  parasites  were  observed.  They 
were  for  the  most  part  round,  though  other  forms  were  also 
"present,  and  from  2  to  4  mikrons  in  diameter.  Most  of  the 
periphery  was  robin's  egg  blue,  the  center  was  unstained. 
Each  of  the  bodies  showed  a  larger  and  a  smaller  lilac-colored 
mass.  Sections  of  the  same  material  showed  the  same  bodies. 
The  evidence  adduced  as  to  the  parasitic  nature  of  the  bodies 
is  insufficient  to  show  that  they  are  not  the  products  of  cell 
degeneration.  Animal  experiments  were  negative. 

*Journ.     Med.  Research.     Vol.  X.,  1904.     pp.  472-482. 


APPENDIX. 


GLOSSARY  OF  TERMS.* 

AGAR  HANGING  BLOCK,  a  small  block  of  nutrient  agar  cut  from  a 

poured  plate,  and  placed  on  a  cover-glass,  the  surface  next  the 

glass  having  been  first  touched  with  a  loop  from  a  young  fluid 

culture  or  with  a  dilution  from  the  same.     It  is  examined  upside 

down,  the  same  as  a  hanging  drop. 
AMEBOID,  assuming  various  shapes  like  an  ameba. 
AMORPHOUS,  without  visible  differentiation  in  structure. 
ARBORESCENT,  a  branched,  tree-like  growth. 
BEADED,  in  stab  of~3treke,  disjointed  or  semi-confluent  colonies  along 

the  line  of  inoculation.' 
BRIEF,  a  few  days,  a  week. 

BRITTLE,  growth  dry,  friable  under  the  platinum  needle. 
BULLATE,  growth  rising  in  convex  prominences,  like  a  blistered  surface. 
BUTYROUS,  growth  of  a  butter-like  consistency. 
CHAINS, 

Short  chains,  composed  of  2  to  8  elements. 

Long  chains,  composed  of  more  than  8  elements. 
CILIATE,  having  fine,  hair-like  extensions,  like  cilia. 
CLOUDY,  said  of  fluid  cultures  which  do  not  contain  pseudozoogloeae. 
COAGULATION,  the  separation  of  casein  from  whey  in  milk.     This 

may  take  place  quickly  or  slowly,  and  as  the  result  either  of  the 

formation  of  an  acid  or  of  a  lab  ferment. 
CONTOURED,  an  irregular,  smoothly  undulating  surface,  like  that  of 

a  relief  map. 

CONVEX,  surface  the  segment  of  a  circle,  but  flattened. 
COPROPHYL,  dung  bacteria. 
CORIACEOUS,  growth  tough,  leathery,  not  yielding  to  the  platinum 

needle.     . 
CRATERIFORM,  round,   depressed,  due    to    the  liquefaction  of   the 

medium. 
CRETACEOUS,  growth  opaque  and  white,  chalky. 

*  Chart  of  Society  of  AmericaiT^BacterioIogists.     Jan.,  1908. 
427 


428  MANUAL    OF    BACTERIOLOGY. 

CURLED,  composed  of  parallel  chains  in  wavy  strands,  as  in  anthrax 

colonies. 
DIASTASIC  ACTION,  same  as  DIASTATIC,  conversion  of  starch  into 

water-soluble  substances  by  diastase. 

ECHINULATE,  in  agar  stroke  a  growth  along  line  of  inoculation,  with 
toothed  or  pointed  margins;  in  stab  cultures  growth  beset  with 
pointed  outgrowths. 

EFFUSE,  growth  thin,  veily,  unusually  spreading. 
ENTIRE,  smooth,  having  a  margin  destitute  of  teeth  or  notches. 
EROSE,  border  irregularly  toothed. 

FILAMENTOUS,  growth  composed  of  long,  irregularly  placed  or  inter- 
woven filaments. 
FILIFORM,  in  stroke  or  stab  cultures  a  uniform  growth  along  line  of 

inoculation. 

FIMBRIATE,  border  fringed  with  slender  processes,  larger  than  fila- 
ments. 

FLOCCOSE,  growth  composed  of  short  curved  chains,  variouly  oriented. 

FLOCCULENT,  said  of  fluids  which  contain  pseudozoogloeae,   i.  e., 

small  adherent  masses  of  bacteria  of  various  shapes  and  floating 

in  the  culture  fluid. 

FLUORESCENT,  having  one  color  by  transmitted  light  and  another  by 

reflected  light. 

GRAM'S  STAIN,  a  method  of  differential  bleaching  after  gentian  violet, 
methyl  violet,  etc.     The  +  mark  is  to  be  given  only  when  the  bacteria 
are  deep  blue  or  remain  blue  after  counterstaining  with  Bismark  brown. 
GRUMOSE,  clotted. 

INFUNDIBULIFORM,  form  of  a  funnel  or  inverted  cone. 
IRIDESCENT,  like  mother-of-pearl.     The  effect  of  very  thin  films. 
LACERATE,  having  the  margin  cut  into  irregular  segments  as  if  torn. 
LOBATE,  border  deeply  undulate  producing  lobes  (see  undulate). 
LONG,  many  weeks,  or  months. 
MAXIMUM  TEMPERATURE,  temperature  above  which  growth  does 

not  take  place. 
MEDIUM,  several  weeks. 

MEMBRANOUS,  growth  thin,  coherent,  like  a  membrane. 
MINIMUM  TEMPERATURE,  temperature  below  which  growth  does 

not  take  place. 
MYCELIOID,  colonies  having  the  radiately  filamentous  appearance  of 

mold  colonies. 
NAPIFORM,  liquefaction  with  the  form  of  a  turnip. 


APPENDIX.  429 

NITROGEN  REQUIREMENTS,  the  necessary  nitrogenous  food.  This 
is  determined  by  adding  to  nitrogen-free  media  the  nitrogen  compound 
to  be  tested. 

OPALESCENT,  resembling  the  color  of  an  opal. 
OPTIMUM  TEMPERATURE,  temperature  at  which  growth  is  most 

rapid. 
PELLICLE,  in  fluid  bacterial  growth  either  forming  a  continuous  or  an 

interrupted  sheet  over  the  fluid. 
PEPTONIZED,  said  of  curds  dissolved  by  trypsin. 
PERSISTENT,  many  weeks  or  months. 
PLUMOSE,  a  fleecy  or  feathery  growth. 

PSEUDOZOOGLOEAE,  clumps  of  bacteria,  not  dissolving  readily  in 
water,  arising  from  imperfect  separation,  or  more  or  less  fusion  of  the 
components,  tut  not  having  the  degree  of  compactness  and  gela- 
tinization  seen  in  zoogloeae. 

PULVINATE,  in  the  form  of  a  cushion,  decidedly  convex. 
PUNCTIFORM,  very  minute  colonies,  at  the  limit  of  natural  vision. 
RAISED,  growth  thick,  with  abrupt  or  terraced  edges. 
RHIZOID,  growth  of  an  irregular  branched  or  root -like  character,  as 

in  B.  mycoides. 
RING,  Same  as  RIM,  growth  at  the  upper  margin  of  a  liquid  culture, 

adhering  more  or  less  closely  to  the  glass. 
REPAND,  wrinkled. 
RAPID,  Developing  in  24  to  48  hours. 

SACCATE,  liquefaction  the  shape  of  an  elongated  sack,  tubular,  cylin- 
drical. 
SCUM,  floating  islands  of  bacteria,  an  interrupted  pellicle  or  bacterial 

membrane. 

SLOW,  requiring  5  or  6  days  or  more  for  development. 
SHORT,  applied  to  time,  a  few  days,  a  week. 
SPORANGIA,  cells  containing  endospores. 
SPREADING,  growth  extending  much  beyond  the  line  of  inoculation, 

i.  e.,  several  millimeters  or  more. 
STRATIFORM,  liquefying  to  the  walls  of  the  tube  at  the  top  and  then 

proceeding  downwards  horizontally. 

THERMAL  DEATH-POINT,  the  degree  of  heat  required  to  kill  young 
fluid  cultures  of  an  organism  exposed  for  10  minutes  (in  thin-walled 
test-tubes  of  a  diameter  not  exceeding  20  mm.)  in  the  thermal  water- 
bath.  The  water  must  be  kept  agitated  so  that  the  temperature 
shall  be  uniform  during  the  exposure. 


43° 


MANUAL    OF    BACTERIOLOGY. 


TRANSIENT,  a  few  days. 

TURBID,  cloudy  with  flocculent  particles;  cloudy  plus  flocculence.. 

UMBONATE,  having  a  button-like,  raised  center. 

UNDULATE,  border  wavy,  with  shallow  sinuses. 

VERRUCOSE,  growth  wart-like,  with  wart-like  prominences. 

VERMIFORM-CONTOURED,  growth  like  a  mass  of  worms,  or  intes- 
tinal coils. 

VILLOUS,  growth  beset  with  hair-like  extensions. 

VISCID,  growth  follows  the  needle  when  touched  and  withdrawn, 
sediment  on  shaking  rises  as  a  coherent  swirl. 

ZOOGLOEAE,  firm  gelatinous  masses  of  bacteria,  one  of  the  most 
typical  examples  of  which  is  the  Streptococcus  mesenterioides  of  sugar 
vats  (Leuconostoc  mesenterioides),  the  bacterial  chains  being  sur- 
rounded by  enormously  thickened  firm  covering,  inside  of  which 
there  may  be  one  or  many  groups  of  the  bacteria. 

NOTES. 

(1)  For   decimal   system   of   group   numbers   see   Table   I.     This 
will  be  found  useful  as  a  quick  method  of  showing  close  relationships 
inside  the  genus,  but  is  not  a  sufficient  characterization  of  any  organism. 

(2)  The  morphological  characters  shall  be  determined  and  described 
from  growths  obtained  upon  at  least  one  solid  medium  (nutrient  agar) 
and  in  at  least  one  liquid  medium  (nutrient  broth).     Growths  at  37°  C 
shall  be  in  general  not  older  than  24  to  48  hours,  and  growths  at  20°  C.  not 
older  than  48  to  72  hours.     To  secure  uniformity  in  cultures,  in  all  cases 
preliminary  cultivation   shall  be  practised  as  described  in  the  revised 
Report  of  the  Committee  on  Standard  Methods  of  the  Laboratory  Sec- 
tion of  the  American  Public  Health  Association.     1905. 

(3)  The  observation  of  cultural  and  bio-chemical   features  shall 
cover  a  period  of  at  least  15  days  and  frequently  longer,  and  shall  be 
made  according  to  the  revised  Standard  Methods  above  referred  to. 
All  media  shall  be  made  according  to  the  same  Standard  Methods. 

(4)  Gelatin  stab  cultures  shall  be  held  for  6  weeks  to  determine 
liquefaction. 

(5)  Ammonia  and  indol  tests  shall  be  made  at  end  of  tenth  day, 
nitrite  tests  at  end  of  fifth  day. 

(6)  Titrate  with  ^  NaOH,  using  phenolphthalein  as  an  indicator: 
make  titrations  at  same  times  from  blank.     The  difference  gives  the 
amount  of  acid  produced. 


APPENDIX.  423 

3.  ENDOSPORES. 

Form,  round,  elliptical,  elongated. 

Limits  of  Size 

Size  of  Majority 

Wall,  thick,  thin 

Sporangium  wall,  adherent,  not  adherent. 

Germination,  equatorial,  oblique,  polar,  bipolar,  by  stretching. 

4.  Flagella    No ..Attachment   polar,    bipolar,   peritrichiate. 

How  stained 

5 .  Capsules,  present  on 

6 .  Zoogloea,  Pseudozoogloea. 

7.  Involution  Forms,  on in days  at °C. 

8 .  Staining  Reactions. 

1:10  watery  fuchsin,  gentian  violet,  carbol  fuchsin, 
Loeffler's   alkaline   methylene   blue. 
Special  Stains 

Gram Glycogen 

Fat Acid   fast 

Neisser 

II.  CULTURAL  FEATURES  (*) 

1 .  Agar  Stroke. 

Growth,  invisible,  scanty,  moderate,  abundant. 

Form  of  growth,  filiform,  echinulate,  beaded,  spreading,  plumose, 

arborescent,  rhizoid. 

Elevation  of  growth,  flat,  effuse,  raised,  convex. 
Lustre,  glistening,   dull,   cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 
Optical    Characters,    opaque,    translucent,    opalescent,    iridescent. 

Chromogenesis   (8) 

Odor,    absent,    decided,    resembling 

Consistency,  slimy,  butyrous,  viscid,  membranous,  coriaceous,  brittle. 
Medium,  grayed,  browned,  reddened,  blued,  greened. 

2.  Pototo. 

Growth  scanty,  moderate,  abundant,  transient,  persistent. 

Form   of  growth,  filiform,  echinulate,  beaded,   spreading,  plumo.se, 

arborescent,  rhizoid. 

Elevation  of  growth,  fiat,  effuse,  raised,  convex. 
Lustre,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

28 


434  MANUAL    OF    BACTERIOLOGY. 

Chromogenesis  (8) Pigment  in  water  insol- 
uble, soluble;  other  solvents 

Odor,    absent,   decided,   resembling 

Consistency,  slimy,  butyrous,  viscid,  membraneous,  coriaceous,  brittle. 
Medium,  grayed,  browned,  reddened,  blued,  greened. 

3 .  Loeffler's  Blood-serum. 

Stroke   invisible,    scanty,    moderate,    abundant.     Form    of    growth, 
filiform,  echinulate,  beaded,  spreading,  plumose,  arborescent,  rhizoid. 
Elevation  of  growth  flat,  effuse,  raised,  convex. 
Lustre,  glistening,  dull,  cretaceous. 
Topography,  smooth,  contoured,  rugose,  verrucose. 

Chromogenesis  (8) 

Medium  grayed,  browned,  reddened,  blued,  greened. 

Liquefaction    begins    in d,     complete    in d. 

4.  Agar  Stab. 

Growth  uniform,  best  at  top,  best  at  bottom;  surface  growth  scanty, 

abundant;  restricted,  wide-spread. 

Line    of    puncture,    filiform,    beaded,    papillate,    villous,    plumose, 
arborescent;    liquefaction. 

5 .  Gelatin  Stab. 

Growth  uniform,  best  at  top,  best  at  bottom. 

Line  of  puncture,  filiform,  beaded,  papillate,  villous,  plumose, 
arborescent. 

Liquefaction,  crateriform,  napiform,  infundibuliform,  saccate,  strati- 
form; begins  in d,  complete  in d. 

Medium   -fluorescent,   browned, 

6 .  Nutrient  Broth. 

Surface  growth,  ring,  pellicle,  flocculent,  membraneous,  none. 
Clouding  slight,  moderate,  strong;  transient,  persistent;  none;  fluid 

turbid. 

Odor,  absent,  decided,  resembling 

Sediment,  compact,  flocculent,  granular,  flaky,  viscid  on  agitation, 

abundant,  scant. 

7.  Milk. 

Clearing  without  coagulation. 
Coagulation    prompt,    delayed,    absent. 

Extrusion  of  whey  begins  in days. 

Coagulum  slowly  peptonized,  rapidly  peptonized. 

Peptonization  begins  on d,  complete  on , d. 

Reaction,  id ,  2d ,  4d ,  lod ,  2od . . 


APPENDIX.  435 

Consistency,  slimy,  viscid,  unchanged. 
Medium  browned,  reddened,  blued,  greened. 
Lab  ferment,  present,  absent. 

8.  Litmus  Milk. 

Acid,  alkaline,  acid  then  alkaline,  no  change. 

Prompt  reduction,  no  reduction,  partial  slow  reduction. 

9.  Gelatin  Colonies. 
Growth  slow,  rapid. 

Form,  punctiform,  round,  irregular,  ameboid,  mycelioid,  filamentous, 

rhizoid. 
Elevation,    flat,     effuse,     raised,     convex,     pulvinate,     crateriform 

(liquefying}. 
Edge,  entire,  undulate,  lobate,  erose,    lacerate,  fimbriate,  filamentous, 

floccose,  curled. 
Liquefaction,  cup,  saucer,  spreading. 

10 .  Agar  Colonies. 

Growth  slow,  rapid,  (temperature ) 

Form,  punctiform,  round,  irregular,  ameboid,  mycelioid,  filamentous, 

rhizoid. 

Surface  smooth,  rough,  concentrically  ringed,  radiate,  striate. 
Elevation,  flat,  effuse,  raised,  convex,  pulvinate  umbonate. 
Edge,    entire,    undulate,    lobate,    erose,   lacerate,  fimbriate,  floccose 

curled. 
Internal  structure,    amorphous,  finely-,  coarsely-granular,  grumose, 

filamentous,   floccose,    curled. 

11.  Starch  Jelly. 
Growth,  scanty,  copious. 

Diastasic  action,  absent,  feeble,  profound. 
Medium  stained 

12.  Silicate  Jelly  (Fermi's  Solution). 
Growth    copious,    scanty,    absent. 
Medium  stained 

13.  Cohn's  Solution. 

Growth  copious,  scanty,  absent. 
Medium  fluorescent,  non-fluorescent. 

14.  Uschinsky's  Solution. 
Growth  copious,  scanty,  absent. 
Fluid  viscid,  not  viscid. 

15 .  Sodium  Chloride  in  Bouillon. 

Per  cent,  inhibiting  growth 

16.  Growth   in    Bouillon   over    Chloroform,   unrestrained,   feeble, 

absent. 

17.  Nitrogen.     Obtained    from  peptone,    asparagin,   glycocoll,    urea, 
ammonia  salts,  nitrogen. 


436  MANUAL    OF    BACTERIOLOGY. 

18.  Best  media  for  long-continued  growth... 

19.  Quick  tests  for  differential  purposes 


III.  PHYSICAL  AND  BIOCHEMICAL  FEATURES. 


i.  Fermentation-Tubes  containing  peptone- 
water  or  Sugar -free  bouillion  and 


g  peptone- 
n  and 

Dextrose 

Saccharose 

rt 

Maltose 

_c 

0 

Mannit 

Gas   production,    in   per   cent. 


H 


Growth     in     closed     arm 


I    I    I    I    !    I    I    I    I    I 


Amount    of   acid    produced    id. 


Amount   of   acid   produced    2d. 
Amount   of   acid   produced   40!. 


Ml 


III       Mill! 


10 
II 

12 


Ammonia   production,   feeble,    moderate,   strong,  absent,   masked 

by  acids. 

Nitrates   in   nitrate   broth. 

Reduced,  not  reduced. 

Presence    of    nitrites ammonia 

Presence   of   nitrates free   nitrogen 

Indol  production,  feeble,  moderate,  strong. 
Toleration  of  Acids:     great,  medium,  slight. 

Acids  tested 

Toleration  of  Na  O  H :  great,  medium,  slight. 

Optimum  reaction  for  growth  in  bouillon,  stated  in  terms  of 

Fuller's  scale 

Vitality  on  culture  media:     brief,  moderate,  long. 

Temperature  relations: 

Thermal  death-point  (10  minutes  exposure  in  nutrient  broth  when 

this  adapted  to  growth  of  organism) C. 

Optimum  temperature  for  growth C.;    or  best  growth 

at  15°  C.,  20°  C.,  25°  C.,  30°  C.,  37°  C.,  40°  C.,  50°  C.,  60°  C. 

Maximum  temperature  for  growth C. 

Minimum  temperature  for  growth C. 

Killed  readily  by  drying:     resistant  to  drying. 

Per  cent,  killed  by  freezing  (salt  and  crushed  ice  or  liquid  air)  ..... 

Sunlight:     Exposure  on  ice  in  thinly  sown  agar  plates:  one-half 

plate  covered  (time  15  minutes),  sensitive,  not  sensitive. 

Per  cent,  killed . . 


APPENDIX. 


437 


13 .  Acids  produced 

14.  Alkalies  produced 

15 .  Alcohols 

16.  Ferments;    pepsin,    trypsin,    diastase,    inverlase,    pectase,    cytase, 
tyrosinase,  oxidase,   peroxidase,  lipase,   catalase,  glucase,   galactase, 
lab.  etc. . 


1 7 .  Crystals  formed : 

18.  Effect  of  germicides: 


Substance 

Method  used 

Minutes 

Temperature 

Killing  quantity 

Amt.  required  to, 
restrain  growth^ 

- 

* 

IV.    PATHOGENICITY. 

1 .  Pathogenic  to  Animals. 

Insects,  crustaceans,  fishes,  reptiles,  birds,  mice,    rats,  guinea-pigs, 
rabbits,  dogs,  cats,  sheep,  goats,  cattle,  horses,  monkeys,  man 

2.  Pathogenic  to  Plants: 


3.  Toxins,  soluble,  endotoxins. 

4.  Non-toxin  forming. 

5 .  Immunity  bactericidal. 

6.  Immunity  non-bactericidal. 

7.  Loss   of  virulence   on   culture   media:    prompt,  gradual,  « 
observed  in months. 


438                                MANUAL    OF    BACTERIOLOGY. 

BRIEF  CHARACTERIZATION. 

Mark  +  or  O,  and  when  two  terms  occur  on  a  line  erase  the  one 
which  does  not  apply  unless  both  apply. 

(2) 
MORPHOLOGY 

Diameter  over  i  /*. 

Chains,  filaments 

Endospores 

Capsules 

Zooglcea,  Pseudozooglcea 

Motile 

Involution  forms 

Gram's  Stain 

(3) 
CULTURAL  FEATURES 

r& 

s 

PQ 

Cloudy,  turbid 

Ring 

Pellicle 

Sediment 

1 

v 

cfl 
S 

*<U 

O 

Shining 

Dull 

Wrinkled 

Chromogenic 
Round 



Proteus-like             • 

Rhizoid 

Filamentous 

Curled 

- 

!_;  ,& 

<U    Cj 

0£ 

Surf  ace  -growth 

Needle-growth 

1 

£ 

Moderate,  absent 

Abundant 

Discolored 

Starch  destroyed 

Grows  at  37°  C. 

Grows  in  Conn's  Sol. 

Grows  in  Uschinsky's  Sol. 

APPENDIX. 


BRIEF  CHARACTERIZATION.— Continued. 


439 


BIOCHEMICAL  FEATURES 

Liquefaction 

Gelatin    (*) 

Blood-serum 

Casein 

Agar,  mannan 

M 

.1 

Acid  curd 

Rennet  curd 

Casein  peptonized. 

Indol  (s) 

Hydro^ 
Ammo 

*en  sulphide 

nia  (s) 

Nitrates  reduced  (s) 

Fluorescent 

Luminous 

DISTRIBUTION 

I 

Animal  pathogen,  epizoon 

Plant 

pathogen,  epithyte 

Soil 

Milk 

Fresh 

water 

Salt  water 

Sewage 

Iron  bacterium 

Sulphur  bacterium 

440  MANUAL    OF    BACTERIOLOGY. 


Surface  divided  in  square  centimeters  for  counting  colonies. 


APPENDIX. 


441 


Jeffer's  plate  (Bausch  and  Lomb).     For  counting  colonies  of  bacteria  on 
circular  plates.     The  area  of  each  division  is  one  square  centimeter. 


442 


MANUAL    OF    BACTERIOLOGY. 


Ol  6 

Plate  for  counting  colonies  of  bacteria  in  Petri  dishes. 


INDEX. 


Abbe  condenser,  20 

Abrin  compared  with  bacterial  toxins, 

189 

immunity  from,   acquired  by  in- 
jections of,  209 

Abscesses,  bacteria  which  cause,  284 
pathological   process   involved, 

282 
metastatic,  caused  by  S.  pyogenes 

aureus,  290 
pathological    process   involved, 

2*90 

Absorbent   cotton   for   plugging   test- 
tubes,  76 
saturated  with  pyrogallic  acid  for 

anaerobic  cultures,  91 
Accidental     infection     of     laboratory 

workers,  115 

Acid,    acetic,  for    decolorizing    back- 
ground of  cover-glass  prep- 
arations, 29 
for  tissues,  41 

as   product  of  growth  of   bac- 
teria, 131 

in  Welch's  capsule  stain,  49 
alcohol     for     decolorizing     after 

stains  for  tubercle  bacilli,  35 
aniline  dyes  which  are  not  suitable 

for  bacterial  stains,  28,  35 
boric  as  an  antiseptic,  257 
butyric,     formation     of     by     B. 

butyricus,  270 
by  various  bacteria,  131 
carbolic,  as  a'germicide,  250 
as  end-product  in  the  growth  of 

bacteria,  129 
germicidal  power  enhanced  by 

hydrochloric  acid,  251 
neutralized    by  sodium  sul- 
phate, 245 

neutralized  by  lime,  251 
in  Ziehl's  carbol-fuchsin  stain, 


34 


Acid,  formic,  as  a  product  of  bacterial 

growth,  131 
fuchsin  not  suitable  for  bacterial 

stains,  28,  35 

hydrochloric,  with  carbolic  acid  as 
germicide,  251 
with    corrosive    sublimate    for 

disinfecting  purposes,  249 
as  germicide   in  gastric  juice, 

164 
hydrochloric-alcohol    as    decolor- 

izer,  35 
lactic,    as    product    of    bacterial 

growth,  131 
in  sour  milk,  157 
formed  by  B.  acidici  lactici  and 
other    bacteria    of  the  same 
group,  131 
picric,  as  contrast  stain  for  tissues, 

28 
propionic,    formed    by    bacterial 

growth,  131 

pyrogalic,    for    cultivating    anae- 
robes, 91 
rosolic,     for    detection    of    acid 

formed  in  cultures,  73 
sulphuric,   in  Gabbett's  decolor- 

izer,  35 
as  decolorizer  with  other  stains, 

Acid-proof  bacilli,  branching  forms  in 

suppurative  and  necrotic  lesions 

and  in  bronchopneumonia,  370 

in  butter,  in  cow   dung   and   on 

grass,  351 

in  gangrene  of  the  lung,  351 
in  milk,  151 

in  smegma  preputialis,  33,  163 
resist  decolorization  with  acids,  33 
retain  the   stain  after  treatment 

with  Gabbett's  solution,  36 
Acids,  formation  of,  by  bacteria,  131 
Acquired  immunity,  200 


443 


444 


INDEX. 


Actinomy.ces,     higher    bacteria     with 
branching  filaments,  277 

in  pus  formation,  284 

tubercle  bacillus  possibly  of  this 
group,  287 

as  the  cause  of  "lump-jaw"   in 

cattle,  actinomycosis  bovis,  366 

Actinomycosis  bovis,  or  "lump-jaw," 

in  cattle,  366 
Active  immunity,  211 
Acute  miliary  tuberculosis,  358 
Adherence  of  bacteria  to  moist  sur- 
faces, 80 

Aerobic  bacteria,  definition  of,  127 
Aerobioscope,  138 
Agar-agar,  azolitmin,  71 

glycerin,  70 

dextrose,  71 

lactose,  71 

litmus,  71 

neutral-red,  71 

plain,  69 

saccharose,  71 

Age,  relation  of,  to  infection,  179 
Agglutinating    substances    in    blood- 
serum,  190 
Agglutinins,  definition,  190 

development  of,  in  blood,  191 

group,  190 

specific,  191 
Aggressins,  217 
Air,  bacteria  of,  137 

B.  anthracis  in,  137 

effect  of  altitude    on    number   of 
bacteria  in,  137 

method  of  examination,  137 

pathogenic  bacteria  conveyed  by, 

J75 

bacteria  on  floating  particles  of 

sputum  in,  137 
conditions  affecting  number  of 

bacteria  in,  137 
species  of  bacteria  in,  137 
in  veins  found  after  death  due  to 

fermentation,  6,  268 
Albumen,  culture  media  containing,  75 
glycerin      fixative      for      tissues 

(Mayer),  40 

specific  precipitins  for,  196 
Alcohol,   acid  for   decolorizing,   after 
staining    bacteria  with    carbol 
fuchsin,  35 

fixation  of  tissues  with,  37 
in  Gram's  stain.  31 
in  Ziehl's  stain,  34 


Alcohol,    relation   to   infection    when 

used  as  beverage,  i 79 
Alexins,  226 

Alimentary  canal,  bacteria  of,  164 
Alum  as  a  coagulant  in  water  filtration, 

141 

Amboceptor,  223,  230 
Ameba  beutali,  417 

bovis,  417 

buccalis,  417 

cobayae,  415 

coli,  415 

coli  felis,  417 

coli  mitis,  417 

dysenterica,  417 

entameba  hominis,  417 

enterica,  417 

fecalis,  417 

gallopavonis,  416 

gemmipara,  417 

gingivalis,  417 

hominis,  416 

intestinalis,  416 

intestini  vulgaris,  417 

Kartulis,  417 

lobosa  coli,  417 

miurai,  417 

muris,  416 

musculi,  416 

pulmonalis,  417 

ranae,  416 

ranarum,  417 

undulans,  417 

urogenitalis,  417 
Amebae,  cultivation  of,  411 

hanging-plate  method,  413 

isolation  of,  412 

permanent  preparations  of,  415 

reproduction  in,  413 

sporeformation,  414 
American  filtration  system,  141 
American  Public  Health  Association, 
directions    for    preparation    of 
culture  media,  66 
Ammonia,  conversion  of,  into  nitrous 

acid,  131 
Anaerobic  bacteria,  cultivation  of,  90 

definition  of,  127 
Anaphylaxis,  211 
Anilin  dyes,  acid  unsuited  for  staining 

bacteria,  28 
alkaline,  for  staining  bacteria, 

28 

alcoholic  solutions  for  prepara- 
tion of  aqueous  solutions,  28 


INDEX. 


445 


Anilin    dyes,    aqueous    solutions    for 

staining  bacteria,  28 
as  germicides,  251 
oil,     aqueous     solution     of,     for 

Gram's  stain,  30,  42 
for  Weigert's  stain,  30,  42 
Animals,  autopsies  on,  104 
care  of,  104 

inoculation  of,  as  means  of  obtain- 
ing pure  cultures,  94 
for  testing  pathogenic  proper- 
ties of  bacteria,  102 
Anopheles  as  conveyers  of  malaria,  177 
as   hosts   in  which   the   malarial 

parasite  develops,  418 
Anthrax  bacillus  (see  B..anthracis) 

symptomatic,    or   "black-leg,"  of 

cattle,  virus  for,  202 
Antiagglutinins,  199 
Antibodies,  199 

Antilysins  analogy  to  antitoxins,  199 
combining    affinities    with    com- 
plement   and    immune    body, 
228  - 
Antiprecipitins  analogy  with  antitoxin, 

199 

Antiseptic,  definition  of,  238 
Antitoxic  unit  in  diphtheria,  348 
Antitoxins   analogy   with   other   anti- 
bodies, 199 
behavior    of    mixtures   of,     with 

toxin,  223 
for  diphtheria,  principle  involved 

in  the  production,  209 
for  tetanus,  331 

method  employed  in  the  pro- 
duction, 346 
Antitoxin-toxin      mixtures,      peculiar 

behavior  of,  223 
Argentamin  as  germicide,  250 
Argonin  as  germicide,  250 
Argyrole  as  germicide,  250 
Arnold's  steam  sterilizer,  55 
Arrhenius,  on  analogy  between  anti- 
toxin-toxin mixtures   and   esters, 

226 
Arrow-poison,   tetanus  bacillus  in,  6, 

22 

Arthritis,  pyogenic  cocci  in,  290 
micrococcus  lanceolatus  in,  306 
gonococcus  in,  314 
Arthrospores,  123 
Artificial  cultivation  of  bacteria,  effect 

of,  on  virulence,  82 
Asiatic  cholera,  396 


Aspergillus  glaucus,  279 
Autoclave,  60 
Auto-infection,  177 
Autopsies    on    animals,     precautions 
against  accidental  infection  at, 

104 

cultures  and  cover-glass  prepa- 
rations from  the  organs, 
105 

technic  employed,  104 
on  human  cadavers,  109 
Avenues  of  entrance  of  bacteria  into 

the  body,  172 
Avian  tuberculosis,  362 

Babes-Ernst  bodies,  121 
Bacilli,    branching   forms    rarely   en- 
countered, 119 
acid-proof,  in  butter  and  on  grass, 

351 

in  gangrene  of  the  lung,  351 
in  milk,  151 

in  smegma  preputialis,  33,  163 
retain  the  stain  after  treatment 

with  Gabbett's  solution,  36 
tubercle  bacilli,  36 
tubercle,  leprosy  and     other 

bacilli,  33 

Bacillus  acidi  lactici,  Hueppe,  273 
acidophilus,  165 
lactis  aerogenes  in  upper  part  of 

intestines  of  children,  164 
morphology  and  cultural  char- 
acters of,  386 
aerogenes  capsulatus,  325 
amylobacter  in  the  stomachs  of 

ruminants,  270 
description  of,  270 
anthracis,     cultivation    in    large 
amounts    for    extraction    of 
endotoxin     susceptible     ani- 
mals, 333 
early  observations    of,    in    the 

blood  of  animals,  14 
formation  of  spores  in  oxygen, 

332 

in  air,  137 
infection  of  laboratory  workers, 

334 

in  soil,  135 
in  "wool-sorter's"  disease,  137, 

334 

morphology,  cultural  charac- 
ters, pathogenic  properties, 

333 


446 


INDEX. 


Bacillus  anthracis,  predisposing  causes 

in  immune  animals,  179 
rapidity    of    distribution    in    the 

animal  on  inoculation,  174 
resistance  of  spores  to  carbolic 

acid,  332 

to  corrosive  sublimate,  333 
to  heat,  332 

spores  present  in  the  soil,  135 
"vaccines"  against,  334 
bifidus  in  human,  feces,  165 
botulinus  in  decayed  meat  and 

other  foods,  159 
buccalis  maximus,  277 
butyricus,  Hueppe,  description  of, 

270 
sive  amylobacter,  Prasmowski, 

270 

capsule  of  Pfeiffer,  315 
capsulatus  septicus,  306 
coli  communis  in  milk,  152,  159 
cystitis  due  to,  290 
detection  of,  in  water,  146 
differentiation  from  B.   typho- 

sus,  382 
in  healthy  intestines  of  man  and 

other  animals,  165 
in  intestines  of  infants,  164 
morphology,    cultural    charac- 
ters,  381 

peritonitis  due  to,  288 
pus  formation  due  to,  284 
resemblance  to  B.  pneumonias 

(Friedlander),  315 
resisting  power  of  toxin  of,  to 

heat,  190 

significance  of,  in  water,  146 
comma,   of  Asiatic   cholera   (see 
Spirillum  cholerae  Asiaticae) 
cyanogenus,  273 
definition  of  the  word,  3 
various  forms,  4 
diphtheriae,    accidental    infection 

of  laboratory  workers,  115 
antitoxin  unit,  348 
found  on  the  bed  clothing  of 

patient,  174 

morphology,    cultural    charac- 
ters, 338,  342 

pathogenic  properties  for  exper- 
iment animals,  344 
preparation    of    the    antitoxin, 

346 

staining  peculiarities,  338 
theories  of  immunity  from,  212 


Bacillus   diphtheriae,   toxin-antitoxins 

mixtures,  223 

enteritidis,    Gartner,    in    decom- 
posed meats  and  other  foods, 

I59 
related   to   paracolon  bacillus, 

386 

erythrosporus,  273 
fluorescens  liquefaciens,  269 

putidus,  269 
fusiformis,  275,  349 
icteroides,  171 
Indicus,  269 
influenza?,  336 
hemorrhagic  septicemia  (Howard, 

316 

Klebs-Loffler  (see  Bacillus  diph- 
theriae) 

leprae,  staining  reaction  like  that 
of  tubercle  bacilli,  33 
description,  morphology,  patho- 
genic properties,  362 
malleic  agglutination  with  homol- 
ogous serum,  193 
morphology,  cultural  characters, 

pathogenic  properties,  364 
magaterium,  270 
mesentericus     vulgatus     (potato 
bacillus),    for   class   demon- 
stration, 114 
high  resisting  power  of  spores, 

123 
morphology,cultural  characters, 

271 
mucosus  (Blumer),  316 

capsulatus  (see  Bacillus  Pneu- 
moniae,  Friedlander) 
mycogenes,    cultural    characters, 
morphology,  pathogenic  proper- 
ties, 317 

mycoides  (B.  ramosus),  271 
Neopolitanus  (probably  the  same 

as  B.  coli  communis,  q.  v.) 
cedematis  maligni  cultural  char- 
acters, 327 

of  blue  milk  (see  Bacillus  cyano- 
genus), 273 
in  soil,  135 

of  bubonic  plague,  320 
of  chancroid,  314 
of  diphtheria  (see  Bacillus  diph- 
theriae) 

of  Ducrey  (see  of  chancroid) 
of  dysentery  (see  Bacillus  dysen- 
teriae) 


INDEX. 


447 


Bacillus  of  Eberth  (see  Bacillus  typhi 

abdominalis) 
of  Emmerich   (see   Bacillus  coli 

communis) 
of   Escherich    (see   Bacillus   coli 

communis) 

of  Friedlander  (see  Bacillus  pneu- 
monias) 

of  glanders  (see  Bacillus  mallei) 
of  influenza   (see   Bacillus   influ- 
enzas) 

of  leprosy  (see  Bacillus  leprae) 
of  malignant  edema  (see  Bacillus 

oedematis  maligni) 
of  ozena,,  probably  same  as  B. 

pneumonias,  g.  v.) 
of  pertussis,  337 
of  rhinoscleroma,  316 
of  Shiga  (see  Bacillus  dysenteriae) 
of  smegma,  33,  143 
of    soft   chancre  (see  Bacillus  of 

chancroid) 
of  syphilis,  Lustgarten,  171 

Joseph  and  Piorkowsky,  171 
of  tetanus  (see  Bacillus  tetani) 
of  typhoid  fever  (see  Bacillus 

typhi  abdominalis) 
of  Vincent,  275,  349 
of  whooping  cough,  337 
of  xerosis,  343 
paracolon,  313 
paratyphoid,  313 
pestis  bubonicae,  320 

cultural  characters,  morphol- 
ogy, pathogenic  properties, 
320 

toxins  from,  190 

vaccine  for,  202 

phlegmones  emphysematosae,  3  26 
phosphorescens  Indicus,  271 
pneumonias,  Friedlander,  cultural 

characters,  morphology,  patho- 
genic properties,  314 

in  suppuration,  284 
prodigiosus  chromogenic  proper- 
ties, 128 

cultural  characters,  morphol- 
ogy, 269 

with     streptococcus     pyogenes 
for  treatment  of  sarcoma,  399 
proteus,  cultural  characters,  mor- 
phology, 219 

in  botulism,  159 

in  suppuration,  284 

mirabitis  vulgaris,  Zenkeri,  319 


Bacillus  pseudodiphtheriae,  343 

pyocyaneus,    cultural   characters, 

morphology,  317 
in  suppuration,  284 
pyogenes   foetidus,    detection    of, 

in  water,  145 

dissemination  by  water,  139 
identity  with  B.  coli  communis, 

38i 

in  suppuration,  284 
ramosus,  (see  B.  mycoides) 
smegmae  preputialis,  33,  36,  163 
subtilis,  271 

tetani,   cultural  characters,   mor- 
phology, 350 
in  soil,  135 

isolation  of,  by  heat,  94 
tuberculosis,    acclimatization    of, 

on  artificial  media,  81,  352 
avain,  362 
in  milk,  151 
in  suppuration,  285 
location  of,  in  animal  tissues, 

352 

morphology,    cultural   charac- 
ters,   pathogenic   properties, 

35° 

of  birds,  362 
peculiar  staining  properties,  33, 

35° 
typhi  abdominalis  (sivetyphosus}, 

addition    of    substances    to 

water  to  facilitate  detection 

of,  147 

contrasted  with  colon,  382 
cultural  characters, morphology, 

37° 
diagnosis  of,  by  Widal  test,  376, 

193 

by  cultures,  371 
infection  of  embryo  with,  172 
in  ice,  148 
in  suppuration,  285 
presence     in    milk    probable, 

!52 

reported     presence    in    water, 

145 

vaginalis  (Doderlein),  163 
violaceus,  270 
Bacteria,  acid-proof,  33,  36 

acclimatization    of,    to    artificial 

media,  81 

adherence  to  moist  surfaces,  80 
aerobic,  127 
anaerobic,  127 


448 


INDEX. 


Bacteria,  antitoxins  for,  200 

diphtheria  antitoxin,  346 

tetanus  antitoxin,  331 
appearance  of,  on  culture  media,  7 
appearance  of  protoplasm  of,  121 
appearance  under  the  microscope 

of,  7 
avenues  of  entry  of,  into  the  body, 

172 
behavior  of,  on  various  media,  81, 

JI3 
capsules  around,  appearance  of, 

49 

apparent  nature  of,  121 
chlorophyll,  relation  to,  i 
chromogenic,  128 
classification  of,  117 
cultivation  of,  94 
coagulation  of  milk  by,  129 
cover-glass  preparations  of,  27,  29 
decomposition  of  cellulose,  129 

of  fat,  129 

of  urine,  129 
definition  of,  3 
diseases  caused  by,  170 
distribution,  4 
early  observation  of,  10 
early  speculations  about,  10 
effects  of  agitation  on,  246 

of  cold  on,  246 

of  desiccation  on,  246 

of  disinfectants  on,  247 

of  electricity  on,  247 

of  heat  on,  125,  247 

of  moisture  on,  126 

of  oxygen  on,  127 

of  preservatives  on,  159 

of  pressure  on,  247 

of  Rontgen  rays  on,  247 

of  sunlight  on,  247 
end-products  of  growth  of,  129 
endotoxins  of,  186 
enzymes  of,  128 
ferments  formed  by,  128 
flagella  on,  125 
fluorescent,  128 
forms  of,  3 
grouping  of,  4,  118 
harmless,  5,  120 
higher,  compared  with  lower,  118 

certain  special,  276 
in  expired  air,  162 
in  feces,  165 
in  fish,  159 
in  stools  of  children,  165 


Bacteiia,  inoculation  of  culture  media 

with,  78 

inversion  of  starch  by,  129 
inversion  of  cane-sugar  by,  129 
involution  forms,  121 
isolation  of,  by  animal  inoculation, 

94 

by  dilution,  94 
by  Esmarch  roll-tube,  99 
by  heating  spore-bearers,  94 
by  plating  in  Petri  dishes,  96 

liquefaction  of  gelatin  by,  128 

method  of  studying,  21,  113 

modification  of  pathogenic  power, 
127 

moisture,  in  relation  to  growth  of, 
126 

morphology  of,  3,  117 

motility  of,  124 

multiplication  of,  122 

nitrifying,  5,  126 

non-pathogenic,  267 

number  of  species  of  non-patho- 
genic, 267 

of  air,  137 

alimentary  canal,  164 

the  conjunctiva,  161,  162 

the  cranial  sinuses,  160 

the  external  genitals,  163 

the  Fallopian  tubes,  160 

the  gall-bladder,  160 

the  ground-water,  139 

the  intestines,  164 

the  mouth,  161 

(B.  buccalis  maximus),  277 

(B.  pneumonias  Friedlander), 

3JS 

(Leptothrix  buccalis),  277 

(Leptothrix  innominata),  277 

(Leptothrix  maxima  buccalis), 
277 

(M.  lanceolatus),  305 

(S.  dentium),  274 

the  nasal  cavity,  161 

the  normal  human  body,  160 

the  urethra,  162 

the  vagina,  162 
part  played  by,  in  nature,  5 
pathogenic,  classification  of,  282 

definition  of,  120 
phosphorescent,  128 
physiology  of,  117 
products    of    growth    of,   acids, 

!3r 
enzymes,  128 


INDEX. 


449 


Bacteria,  products  of  growth  of,  gases, 

I3I 

indol,  129 
pigment,  128 
ptomaines,  153,159 
toxins,  1 86 
pure  cultures  of,  94 
putrefactive,  94 
relation  of  oxygen  to,  127 
resemblance  to  other  living  forms, 

i 
size,  absolute,  4 

variability,  121 
spontaneous    generation    of,  dis- 

proven,  3,  12 
spore  formation,  14 
synonyms  for,  4 
staining  of,  in  cover-slips.  28 
in  sections  of  tissues,  37,  41 
in  sputum,  34,  350 
thermal  death-point  of,  125 
thermophylic,  125 
transmission     of     specimens     by 

mail,  no 
useful,  5,  120 
warmth  in  relation  to  growth  of, 

125 

zooglcea  masses  of,  122 
Bacterial    poisons    in    cheese,    meat, 

milk  and  other  foods,  186 
Bacteriolysis,   by  normal  and  of  im- 
mune blood-serum,  193 
source,  218 

theories  in  regard  to  the  composi- 
tion of  lysins  in  general,  226 
mechanism  of,  230 
Bacterium  acidi  lactici,  316 
aerogenes,  316 
coli  communis  (see  Bacillus  coli 

communis) 
pneumonicum,  316 
syncyanum   (see  Bacillus  cyano- 
genus),  termo,  definition,  134 
(see  also  Bacillus  proteus) 
ureae,  273 
^  Zopfii,  273 
Bail  on  aggressins,  217 
Balsam,  Canada,  for  mounting  cover- 
slip  preparations,  27,  29 
Basic  anilin  dyes,  28 
Basophylic  granules,  41,  43 
Bed-bugs,  diseases  carried  by,  177 
Beggeatoa,  276 
Beef-tea,  64 
Beri-Beri,  170 
20 


Berkefeld  filter,  63 

Bichlorid  of  mercury  as  germicide,  248 

Biedert  method  for  examining  sputum, 

37 

Birds,  tuberculosis  of,  362 
Bismarck  brown  as  contrast  stain  with 
Gram's     method     and     other 
methods,  28,  30 
Black  death,  325 
Black-leg  vaccine,  202 
Blood-agar  for  cultivating  B.    influ- 

enzae,  336 
M.  gonorrheae,  312 
Blood,  cultures  from,  108 
Blood-poisoning,  286 
Blood-serum,  agglutinins  in,  190 
aggressins  in,  217 
effect  of  dilution  upon,  233 
effect  of  heating  on  bacteriolytic, 

potency  of,  195 

of  standing,  on  potency  of,  194 
bactericidal  properties  of,  193 
cytolytic  properties  of,  194 
heterologous  and  homologous, 

197 

inactivated,  195,  222 
Loffler's,  75 
lysins  in,  193 
opsonins  in,  216 
precipitins  in,  196 
preparation  of,  for  culture   me- 
dium, 73 

reactivated,  195,  222 
specific  precipitins  for,  196 
sterilization  of,for  culture  medium, 

59,  74 

test  for  typhoid  fever,  193 
Blue  milk,  bacillus  of   (see  Bacillus 

cyanogenus), 
pus,  318 
vitriol,  258 

Bodily  conditions  predisposing  to  in- 
fection, 178 

Boiling,    sterilization    by,    effect    on 
spores,  247 
efficacy  of,  55 
for  sterilizing  water,  142 
for  surgical  instruments,  266 
Boils,  caused  by  S.  pyogenes  aureus, 

293 

Boophilus  annulatus,  177 
Bordet's  theory  of  nature  of  lysins,  226 
of  toxin-antitoxin  reaction,  225 
on    difference    between    his    and 
Ehrlich's  theories,  229 


45° 


INDEX. 


Bordet's  theory  on  bactericidal  action 
of  ox-serum,  236 

on  dilutions  of  serum,  233 
Boric  acid  (see  Acid,  boric) 
Bouillon,  64 

sugar-free,  67 

Bovine    tuberculosis,    danger    of,    in 
milch  cows,  151 

lesions  of,  355 

Branching  forms  of  bacilli,  119 
Bread-paste,  76 
Bromin  as  a  germicide,  256 
Bronchitis,  B.  influenzae  in,  337 

streptococci  and  pneumococci  in, 

289 

Brownian  movement,  24 
Bubonic  plague,  bacillus  (see  Bacillus 

pestis  bubonicae) 
Buchner's     method     for     cultivating 

anaerobes,  90 
Burner,  Koch's  safety,  86 
Butter,  tubercle  bacilli  in,  151 
Butyric  acid  (see  Acid,  butyric) 

Cadaver,     care      of,     in      contagious 
diseases,  259 

Calcium    compounds    as    germicides, 
256 

Canada   balsam  for  mounting  cover- 
glass  preparations,  27,  29 
for  mounting  sections,  41 

Capaldi's  culture-medium,  347 

Capsule  bacillus  of  Pfeiffer,  315 

Capsules  of  bacteria,  definition  of,  121 
nature  of,   staining  of,   by  Hiss' 
method,  by  Welch's  method,  49 

Carbol-fuchsin    for  staining   tubercle 
bacilli,  34 

Carbolic  acid  (see  Acid,  carbolic) 

Carbon  dioxid,  as  a  product  of  bac- 
terial growth,  132 

Carbuncles    caused    by    S.    pyogenes 
aureus,  293 

Carmine,  as  contrast-stain  in  Gram- 

Weigert's  method,  43 
in  gentian-violet  preparations,  45 

Caries  of  the  teeth,  162 

Carriers  of  infection,  175 

Caseation,  357 

Catgut,  surgical  preparation,  difficulty 
of  sterilizing,  265 

Cedar-wood  oil,  for  immersion-lens,  20 

Celloidin  imbedding,  28 

Cells,  epithelioid,  giant,  357 
pus,  283 


Chlorophy 
affe< 


Cellulitis  caused  by  bacillus  aerogenes 

capsulatus,  326 

by  streptococcus  pyogenes,  296 
Cellulose,  decomposition    by  bacteria 
in  the  stomachs  of  ruminants, 
129,  165 

Centrifuge  for  milk  separator,  154 
Cerebro-spinal  meningitis,  309 
Chancroid,  bacillus  of,  314 
Charbon  (see  Anthrax) 

symptomatique  (see  Symptomatic 
anthrax) 
Cheese-poisoning,  153 

chemical     nature     of     antitoxic 

action,  220 

Chemotaxis,  definition,  125 
in  inflammation,  214 
in  suppuration,  282 
Chicken-pox,  170 
Chlorid  of  lime,  as  germicide,  256 
Chlorin  as  a  germicide,  255 
Chloroform  as  a  preservative  for  blood- 
serum,  74 

>hyll,   absence  of,   in  bacteria 
affects  their  nutrition,  126 
not  present  i%  bacteria,  i,  167 
in  higher  plants,  134 
Cholera,  Asiatic,  diagnosis,  396 
B.  dysenteriae  in,  387 
B.  proteus,  320 
infantum,  320 
nostras,  401 
red  reaction,  392 
spirillum  (see  Spirillum  of  Asiatic 

cholera),  390 

Chromogenic  bacteria,  128 
Cladothrix,  276 
Classes    in    bacteriology,     hints    for 

teaching,  113 

Classification  of  bacteria,  117 
Cleaning  fluid,  25 

Climate,  influence  on  infections,  179 
Clostridium  butyricum,  definition,  124 
Coal-oil  for  destroying  insects,  258 
Cold,  effect  of,  on  growth  of  bacteria, 

124 

Collection  of  material  for  bacterio- 
logical examination  of  sputum, 
of  urine,  107 

in  suspected  diphtheria,  109 
Collection   of   samples   of   water  for 
bacteriological  examination,  142 
Collodion,    capsules,    for    cultivating 
bacteria  inside  the  animal  body, 
106 


INDEX. 


451 


Collodion,  for  imbedding  tissues,  38 
Colon  bacillus  (see  Bacillus  coli  com- 

munis) 

Colonies  of  bacteria,   appearance  of, 
examination  of,  how  obtained, 
100 
Comma     bacillus     of     cholera     (see 

Spirillum  of  cholera) 
shaped  bacteria,  118,  120 
Complement   analogy  with   enzymes, 

229 

diversion  of,  233 
effects  of  heat  on,  195 
mode  of  action  of,  228 
where  found,  232 
Condenser,  Abbe,  20 
Conjunctivitis,  gonorrheal,  314 
Consumption,  355 
Contagious  disease,  definition,  169 
diseases,  disinfection  after,  260 
Contrast  stain,  acid  fuchsin,  28 
Bismarck  brown,  28,  31,  37 
eosin,  28,  31,  48 
for  tubercle  bacilli,  33 
hematoxylin,  44 
picric  acid,  28 
methylene-blue  for  carbol-fuchsin 

preparations,  37 
Copper  sulphate,  258 
Copperas,  258 
Cornet  forceps,  25 
Corrosive  sublimate,  248 
Cotton,  absorbent,  for  anaerobic  cul- 
tures, 91 

as  plugs  for  test-tubes,  76 
power  of  filtering  bacteria  out  of 

air,  13 

Cover-glass  forceps,  26 
Cover-glass    preparations,    fixing    of, 

27 

impression  preparations,  25 

smear  preparations,  26 

staining  of,  28 
Cover-glasses,  cleaning  fluid  for,  25 

handling  of,  25 

Cowpox,  discovery  of  protective  value 
of,  for  small-pox,  n 

used  for  vaccination,  200 
Cranial  sinuses,  bacteria  of,  160 
Cream  ripening,  151 
Creolin,  251 
Cresol,  251 

Croup,  membranous,  345 
Croupous    pneumonia,    diagnosis   of, 
301 


Croupous    pneumonia,    due    to   pus 
cocci,  288 
B.  pneumoniae,  315 
M.  lanceolatus,  305 
Cultivation  of  amebse,  410 

of    bacteria    in    collodion     sacs, 

1 06 

in  test-tubes,  78,  101 
on  plates,  95 
under   anaerobic   conditions:    by 

Buchner's  method,  91 
by  Esmarch's  method,  92 
by  Frankei's  method,  91 
by  Hiippe's  method,  93 
by  Koch's  method,  93 
by  Liborius's  method,  92 
by  Novy's  method,  92 
by  Park's  method,  93 
by  Wright's  method,  91 
of  trypanosomes,  409 
Culture  media,  agar-agar,  plain,  69 
with  blood-serum,  75 
with    dextrose,    with    lactose, 

with  saccharose,  71 
with  glycerine,  70 
blood-serum,  73 
bouillon,  plain,  64 

with    dextrose,    with    lactose, 

with  saccharose,  67 
sugar-free,  64 
Loffler's  mixture,  75 
milk,  72 
nitrate  broth,  73 
object  of  sterilizing,  64 
preparation  of  test-tubes  for,  76 
sterilization  of  blood-serum,  74 
of  ordinary  media,  55,  58 
of  milk,  72 
of  sugar  media,  67 
of  test-tubes  for  culture  media, 

76 
Cultures  at  autopsies,  upon  animals, 

104 

on  human  cadavers,  109 
destruction  of,  116 
from  the  blood,  108 
Cupric  sulphate,  258 
Cutting  sections  of  tissues,  40 
Cystitis,  caused  by  B.  coli,  290 
by  B.  lactis  aerogenes,  386 
B.  proteus  in  urine  in,  320 
Cytolysins,  193 
Cytolysis,  193 

Cytolytic     potency    of    blood-serum, 
194 


452 


INDEX. 


Decomposition,  129 
Delafield's  hematoxylin,  44 
Deneke's  spirillum,  402 
Dengue,  170,  172 
Dental  caries,  162 
Deodorizers,  238 
Dermatitis,  blastomycetic,  281 
Dextrose-agar,  71 
Dextrose-bouillon,  67 
Diagnosis  of  actinomycosis,  368 
of  bubonic  plague,  330 
of  cholera,  396 
of  diphtheria,  297,  338 
of  dysentery  (from  cultures),  387 

(serum  test),  388 
of  glanders  (Straus'  method),  365 
of  gonorrhea,  312 
of  influenza,  337 
of  malaria,  432 
of  Malta  fever,  308 
of  meningitis,  cerebro-spinal,  310 
of  pneumonia,  306 
of  tuberculosis  (tuberculin  injec- 
tion), 261 

(microscopic  examination),  251 
of  typhoid  fever,  -(appearance  of 

culture),  371 
(Gruber-Widal  test),  376 
(from  stools),  378 

Diphtheria,     antitoxin     (behavior    of 

toxin-antitoxin  mixtures),  223 

methods    of    production),  209, 

346 
bacillus  of  (accidental  infection, 

"5 

(cultural  characters  and  mor- 

phology),  338 
(in  throats  of  healthy  persons), 

175 

diagnosis,  338 

toxin  (nature),  187 

(analogy  with  ferments),  189 
(effect  on  animals  on  injection), 

209 

(mixed  with  antitoxin),  223 
(death  due  to),  345 
Diphtheritic  membrane,  location,  345 
Diplococcus,  definition,  119 

intercellularis    meningitidis    (cul- 
tural characters  and  morphol- 
ogy), 3°9 
(in  suppuration),  284 

of  gonorrhea,  311 

of    pneumonia   (see  Micrococcus 
lanceolatus) 


Disease,  bacteria  in,  170 
Diseases  caused  by  bacteria,  170 
by  protozoa,  425 
probably  due  to  microorganisms, 

171 

infectious,  immunity  from,  199 
dissemination   of:    by  fleas,  flies, 

bed-bugs,  176 
by  mosquitoes,  ticks,  177 
Disinfectants,  definition,  239 

effects   of    the    character   of    the 
medium  in  which  the  tests  are 
made  upon,  241 
elective  affinity  of  certain,  241 
methods  of  testing  the  potency  of, 

242 

specific  action  of  certain,  241 
testing  of  the  potency  of  gaseous, 

245 

determination  of  the  potency  of, 
242 

physical,  246 

chemical,  247 
Disinfection  at  autopsies,  259 

definition  of,  238 

of  cadavers   after   infectious   dis- 
ease, 260 

of  dejecta,  258 

of  hands,  264 

of  houses,  260 

of  mouth,  258 

of  sputum,  259 

of  stools,  259 
'   of  urine,  259 

surgical,  252 
Distribution    of    bacteria    in    nature, 

*35 

Diversion  of  complement,  233 

Dorset's  egg-medium,  76 

Dressings,  surgical  preparation,  265 

Drigalsky-Conradi's   method   for   de- 
tecting typhoid  bacilli  in  water, 

374 

Drinking  water,  purification  of,  140 
Drying,    influence    on    bacteria,    (see 
Bacteria,   effect  of  desiccation 
upon) 

Ducrey's  bacillus,  314 
Dunham's  peptone  solution,  73 
Dyes,  aniline,  28 

as  germicides,  251 
for  bacterial  stain,  28 
Dysentery,  ameba  of,  415 
B.  dysenteriae  in,  386 
B.  pyocyaneus  in,  318 


INDEX. 


453 


Ear,  middle,  160,  289 
inflammation  of,  289 
normally  sterile,  160 

Eberth's    bacillus     (see     Bacillus    of 
typhoid  fever) 

Edema,     malignant,      bacillus      (see 
Bacillus  cedematis  maligni) 

Egg-albumen  as  a  culture-medium,  75 

Egg-medium  of  Dorset,  76 

Eggs,  in  cultivating  anaerobes,  75,  82 

Ehrlich's  side-chain  theory,  218 

Electric     heater    and     regulator    for 
incubators  (Rogers),  87 

Electricity,  influence  on  bacteria,  247 

Eisner's  culture-medium,  374 

Embryo,  infection  of,  172 

Emmerich's     bacillus     (see     Bacillus 
coli  communis) 

Emphysematous  gangrene  (see   Bacil- 
lus aerogenes  capsulatus),  325 

Endocarditis,  M.  gonorrhcese  in,  311 
M.  lanceolatus  in,  306 
pyogenic  bacteria  in,  288 
S.  pyogenes  aureus  in,  293 
streptococcus  pyogenes  in,  296 

Endogenous  spores,  122 

Endotoxins,  186 

Enzymes,  analogy  with  toxins,  189 
bacterial,  128 

Eosin  in  Wright's  stain,  48 

Epithelioid  cells,  357 

Epitoxoid,  224 

Epitoxonoid,  224 

Erysipelas  (see  Streptococcus  of    ery- 
sipelas) 

Escherich's  bacillus  (see  Bacillus  coli 
communis) 

Esmarch's  method  for  anaerobes,  92 
roll-tubes,  99 

Essential  oils  as  germicides,  257 

Eye-piece,  18,  19,  20 

Fallopian  tube,  bacteria  of,  160 
Farcy-buds,  365 

Fats,  decomposition  by  bacteria,  129 
Favorable  and  unfavorable  conditions 

for  growth  of  bacteria,  125 
Feces,  bacillus  of  tetanus  in,  329 
bacteria  of,  165 
disinfection  of,  259 
typhoid  bacilli,  examination-  for, 

by  serum  test,  376 
in  water  or  feces,  373 
Fermentation,  12,  133 
tube,  132 


Ferments,  bacterial,  1 28 

and  toxins,  189 
Ferrous  sulphate,  258 
Fibrin,  Weigert's  stain  for,  42,  258 
Picker-Hoffmann's  method  for  detect- 
ing typhoid  bacilli,  374 
Film-preparations,  making  of,  26 

fixing  of,  27 

staining  of,  28 
Filter,  alum,  141 

for  bacteria,  62 

American,  141 

Berkefeld,  62 

infusorial  earth,  62 

Kitasato,  62 

mechanical,  141 

Pasteur-Chamberland,  62,  142 

sand,  141 

unglazed  porcelain,  142 
Filterable  microorganisms,  171 
Filtration,  sterilization  by,  141 

of  water,  63,  140 
Finkler  and  Prior  spirillum,  401 
Fishing  from  colonies,  91 
Fission,  multiplication  of  bacteria  by,  3 
Fixation  of  cover-glass   preparations, 
26,  27,  28 

of  slide-preparations,  27 

of  tissues,  37 

Fixative,  glycerin  albumen,  39 
Flagella,  as  organs  of  locomotion,  125 

staining  of,  50 

Fleas,  diseases  carried  by,  177 
Flies,  cholera  carried  by,  176,  395 

tuberculosis  carried  by,  176 

typhoid  fever  carried  by,  176,  389 
Fluid  for  cleaning,  25 
Fluorescence  of  bacteria,  in  water,  128 

B.  fluoresceis  liquefaciens,  269 

B.  pyocyaneus,  317 
Focusing  the  microscope,  21 

for  hanging-drop  preparations,  23 
Fomites,  definition,  169 
Food  used  by  bacteria,  126 
Foods,  bacteria  of,  149 

poisoning  by,    159 
Foot-and-mouth  disease,  171 
Forceps,  Cornet,  26 

cover-glass,  26 

for  slides,  Kirkbride,  27 

Stewart,  26 
Formaldehyde  as  a  germicide,  251 

candles,  254 

for  disinfection  of  rooms,  260 

fixation  of  tissues  with,  37 


454 


INDEX. 


Formalin,  251 

lamps,  253 

Formic  acid  (see  Acid,  formic) 
Fowl-cholera,    protective    inoculation, 

201 

Fowls,  tuberculosis  of,  362 
Fractional  sterilization,  55 
Frankel's  method  for  anaerobes,  92 
pneumococcus    (see   Micrococcus 

lanceolatus) 

Freeman's  pail  for  pasteurizing,  60 
Freezing,  influence  on  bacteria,  246 
Friedlander's  bacillus  of  pneumonia 

(see  Bacillus  pneumoniae) 
Fuchsin,  as  bacterial  stain  in  general, 

28 

acid  as  contrast-stain,  28 
carbol  for  tubercle  bacilli,  34 
Furbringer's   method  for  disinfecting 

hands,  264 
Fusiform  bacillus  of  Vincent,  275 

Gabbett's  method  for  staining  tubercle 

bacilli,  35 

Gall-bladder,  colon  bacillus  in,  384 
typhoid  bacilli  in,  378 
usually  sterile,  160 

Gangrene,  emphysematous    (see    Ba- 
cillus     aerogenes     capsulatus, 

325 

Gas-burner,  Koch's,  86 
Gas-formation  by  bacteria,  131 
phlegmons,  327 
regulator,     mercurial,     Reichert, 

Roux,  84 

Gastric  juice,  germicidal  power,  164 
Gelatin,  nutrient,  68 
liquefaction,  128 
tetanus  bacilli  in,  329 
Gelose  (see  Agar-agar) 
Gentian- violet,  31 
Geppert's  test  for  germicides,  244 
Germicidal  power  of  blood-serum,  due 

to  lysin,  166 

effect  of  dilution  upon,  233 
inactivated  by  heat,  222 
nature  of,  226 
reactivation  of,  222 
Germicide,  definition,  239 

determination  of  the  potency  of, 

242 

effects  of  the  character  of  the  me- 
dium  in   which   the   tests   are 
made  upon,  241 
elective  affinity  of  certain,  241 


Germicide,    methods    of    testing    the 

potency  of,  242 
specific  action  of  certain,  241 
testing  of  the  potency  of  gaseous, 

245 

Germicides,  tests  for  potency  of,  242 
Germ,  use  of  the  word,  3 
German  measles,  170 
Giant-cell,  357 
Glanders  bacillus,  morphology,cultural 

characters,  364 
Straus's  method    for    diagnosing, 

365 

Glassware,  sterilization  of,  53 
Gloves,  rubber,  265 
Glucose,  agar,  71 

bouillon,  67 
Glycerin-agar,  70 

albumen,  40 

bouillon,  67 

Gonococcus  of  Neisser,  in  suppura- 
tion, 284 

morphology,    cultural   characters, 

3ii 

Gram-Giinther  method,  32 
Gram's  method  for  cover-glass  prep- 
arations, 31 
for  sections,  42 
bacteria  stained  by,  32 

not  stained  by,  32 
Gram-Weigert  method,  42 
Gray  tubercle,  358 

Green  pus  (see  Bacillus  pyocyaneus) 
Ground-water,  139 
Group  agglutinins,  191 
lysins,  194 
precipitins,  197 
Groups  of  bacteria,  118 
Gruber-Widal  reaction,  193 
Guarnieri's  medium,  75 
Gun-cotton,  38 

Haffkine's  inoculations  for  plague, 
active  immunity  produced  by, 
212 

cultures  employed  in,  323 

preparation  of  vaccine,  202 
Hair-follicles,  infection  around,  286 
Hands,  disinfection,  264 
Hanging-block,  24 

drop,  23 

Haptophore,  219 
Hardening  of  tissues,  in    alcohol,    in 

formalin,  37 
Hay  bacillus,  for  class  work,  114 


INDEX. 


455 


Hay  morphology,  cultural  characters, 
271 

resisting  power  of  spore  to  heat, 

123 
Heat,  effect  on  growth  of  bacteria,  247 

sterilization  by,  53 
Hematoxylin,  44 
Hematozoon  of  malaria,  432 
Hemolysis,  194 

Healthy  persons  as  carriers  of  infec- 
tion, 175 

Heterologous  serum,  191 
High  temperature  incubator,  82 
Higher  bacteria,  276 
Hill's  test  for  germicides,  243,  §2 
Hiss,  medium  of,  373 

stain  for  capsules,  49 
Historical  sketch  of  bacteriology,  8 
Hog  cholera,  171 
Holmes,  O.  W.,  12 
Homologous  serum,  191 
Honing  of  knives,  40 
Hot-air  sterilizer,  54 
Houses,  disinfection,  253,  260 
Hueppe's  method  for  anaerobes,  82 
Hydrochloric  acid   (see  Acid,   hydro- 
chloric) 

Hydrogen,    cultivation    of    anaerobes 
under,  8q 

peroxide,  257 

sulphide,  119 
Hydrophobia,  organism,  170 

diagnosis  of,  203,  208 

effect  of  radium  rays  on  virus  of, 
206 

incubation  period  in,  207 

preventive  inoculation,  206 

"virus  fixe,''  206 
Hypersensibility  to  infection,  n 
Hypha,  280 

Hypochlorite  of  calcium,  256 
Hypodermic   inoculation   of   animals, 


Ice,  bacteria  of.  148 
Ice-cream  poisoning,  153 
Illumination  for  the  microscope,  21 
Imbedding,  in  celloidin,  in  paraffin,  38 
Immune-body,  226 
Immunity,  acquired,  200 

active,  211 

antitoxic,  208 

bacteriolytic,  193 

by  injection  of  cultures,  201 

duration  of,  212 


Immunity,  individual,  198 

following  vaccination,  200 
scope  of,  198 
natural  199 
passive,  211 
racial,  199 

side-chain  theory,  218 
theories  of,  212 
unit,  348 

Impression-preparation,  25 
Inactivated  serum,  222 
Incubator,  82 
Indol,   formation  of,  by  bacteria,  1 29 

test  for,  129 

Infected  surgical  wounds,  262 
Infection,  auto-,  177 
avenues  of,  172 
bodily  conditions  favoring,  178 
by  air,  175 
by  bed  bugs,  177 
by  flies,  176,  380 
by  healthy  individuals,  175 
by  insects,  177 
by  mosquitoes,  172,  177 
in  milk,  water,  soil,  176 
influence  of  amount  of  material 

on,  182  i 

of  virulence  on,  182 
local  conditions  favoring,  181 
mixed,  183 
modes  of,  172 
of  embryo,  172 
of  investigators  with  pathogenic 

bacteria,  115 
of  wounds,  263 

predisposing    causes:     Age,   alti- 
tude, climate,  individual,  179 
racial,  180 
secondary,  183,  287 
terminal,  183 
Infectious  disease,  definition,  168 

diseases  not  followed  by  immunity, 

200 
Inflammation,  283,  288 

diphtheritic    (see     Pseudo-mem- 
branous inflammation) 
Influenza  bacillus,  336 
Infusorial  earth  in  filters,  62 
Inoculation   of    animals,    for   testing 
pathogenic  property  of  bac- 
teria, 103 

for  isolation  of  bacteria,  94 
intravenous,  103 
subcutaneous,  102 
of  tube-cultures,  79 


456 


INDEX. 


Inoculations,  preventive,  201 

for  anthrax,  201 

for  black-leg  of  cattle,  16,  202 

for  bubonic  plague,  323 

for  cholera,  394 

for  erysipelas  of  swine,  202 

for  fowl-cholera,  201 

for  hydrophobia,  203 

for  small-pox,  10,  200 

for  tuberculosis,  362 

for  typhoid  fever,  381 
Insects,  destruction  of,  255,  258 

infection   spread  by   (see   Infec- 
tion) 

Instruments,  surgical  preparation,  266 
Intermittent  sterilization,  55 
Intestine,  bacteria  of,  164 
Intravenous     inoculation    of    rabbits, 

103 

Invisible  microbes,  121 
Involution  forms  of  bacteria,  121 
Iodide  of  mercury,  249 
Iodine  solution,  in  Gram's  stain,  31 

in  Weigert's  stain,  41 
lodoform,  257 
Iris  diaphragm,  18 

Isolation  of  bacteria:  by  animal  inoc- 
ulation, by  dilution,  by  heat- 
ing, by  plating,  94 

by  Esmarch  roll-tubes,  99 

historical,  15 
Itch,  15 

Jenner,  n 

Journals  of  bacteriology,  8 

Kerosene,  for  destroying  insects,  258 
Kirkbride  forceps  for  slides,  27 
Kitasato  filter,  62 

Klatschpreparat   (impression  prepara- 
tion), 25 
Klebs-Loffler    bacillus    (see    Bacillus 

diphtherias) 

Knives,  sharpening  of  microtome,  40 
Koch,  discovery  of  B.  tuberculosis  by, 

16 
Koch's  gas-burner,  86 

method  for  anaerobes,  93 
plate-cultures,  15 

as    means  of  isolating  bac- 
teria, 94 
(historical),  15 
use  of,  at  autopsies,  105 
.postulates  for  proving  the  patho- 
genic property  of  bacteria,  168 


Koch's  steam  sterilizer,  59 
tests  for  germicides,  243 

Lactic  acid  (see  Acid,  lactic) 
Lactose,  agar,  71 

bouillon,  67 
Leeuwenhoek,  9 
Leprosy  bacillus,  362 

morphology,  cultural   characters, 
pathogenic  properties,  362 

resemblance    to    B.   tuberculosis, 

351 

Leptothrix  (in  normal  mouth),  161 
(morphology,  cultivation  of),  277 
buccalis,  innommata,  maxima 

buccalis,  277 
Leucin,  as  product  of  bacterial  growth, 

129 
Leucocytosis,  artificial,  215 

in  disease,  214 
Leucomaines,  188 
Ligatures,  surgical  preparation,  265 
Light,  influence  on  bacteria,  247 
Lime  as  a  germicide,  256 
Liquefaction  of  gelatin,  128 

analogy     to     trypsin      digestion, 

128 
as    a    means    of     classification, 

129 

Lister,  14 

Lithium-carmine,  45 
Litmus- agar,  71 

milk,  73 

Lockjaw  (see  Tetanus)* 
Loffler's  bacillus  of  diphtheria  (acci- 
dental   infection    from    cul- 
tures), 115 
(in  throats  of  healthy  persons), 

!75 

(morphology,   cultural    charac- 
ters, pathogenic  properties), 

338 

(see  also  Bacillus  diphtherias) 
blood-serum,  75 
methylene-blue,  30 
stain  for  flagella,  50 
Lump- jaw,  366 
Lungs,  bacteria  of  the,  160 
Lustgarten's  bacillus  of  syphilis,  171 
Lymphoid  tissues,  relation  of  bacteria 

to,  1 60,  173 
Lysins,  nature  of  ,193 
inactivated,  195 
reactivated,  195 
Lysol,  251 


INDEX. 


457 


Macrophages,  212 
Madura  disease,  Madura  foot,  369 
Magnifying  power  of  objectives,  21 
Mails,   transmission  of  specimens  of 

bacteria  in,  no 

Malachite-green  as  a  germicide,  251 
Malaria,  destruction  of  mosquitoes  of, 

with  sulphur  dioxid,  255 
with    petroleum,  258 
mosquitoes  as  carries  of,  177 
parasite  of,  432 
Malarial     parasite,     preparation     of 

blood-films,  1 08 
staining  of,  in  blood-films,  46 
Malignant  edema,  bacillus  in  soil,  135 
morphology,   cultural  characters, 

pathogenic  properties,  327 
pustule  (see  Bacillus  anthracis) 
Mallein,  extract  from  cultures,  190 

method  of  preparation,  366 
Malta-fever,  micrococcus  of,  307 
Marmorek's  antistreptococcus  serum, 

298 
Massachusetts  State  Board  of  Health 

steam  sterilizer,  58 
Mastzellen,  41 

Mayer's  glycerin-albumen,  40 
Measles,  complicated  with  diphtheria, 

246 

microorganism  undiscovered,  170 
streptococcus  in,  298 
Meat,  tubercle  bacilli  in,  152 
Mechanical  filtration  of  water,  141 
Medium,  culture  (see  Culture-medium) 
Membranous  croup,  rhinitis,  345 
Meningitis,  streptococcus  pyogenes  in. 

297 

M.  lanceolatus  in,  306 
diplococcus  intracellularis  menin- 
gitis in,  309 
B.  pneumonias  in,  315 
Mercuric   chloride  (see  Bichloride  of 

mercury), 
iodide,  249 
Mercurol,  250 
Mercury   bichloride   of   mercury   (see 

Bichloride) 
Metachromatic  granules   of   bacteria, 

121 

Metastatic  abscesses,  290 
Metchnikoff,   theory  of  phagocytosis, 

212 

vibrio  of,  400 

Methyl-alcohol  lamp  in  formaldehyde 
disinfection,  254 


Methylene-blue,  as  a  stain  for  bacteria, 

28 

as  a  germicide,  251 
Loffler's,  30 

Methyl- violet  as  a  germicide,  251 
Miasmatic  disease,  definiton,  169 
Microbe,  use  of  the  word,  3 
Micrococcus  agilis,  268 
amylovorus,  168 
definition,  118 

gonorrhoeae,  in  auto-infection,  178 
morphology,    cultural    charac- 
ters, pathogenesis,  311 
lanceolatus  changes  on  artificial 

cultivation,  302 
diseases  caused  by,  306 
habitat,  305 

in  normal  human  mouth,  161 
in  suppuration,  284 
means  of  differentiation  from 

streptococcus,  392 
method  of  staining  capsules  of, 

J31 
morphology, cultural  characters, 

pathogenesis,  301 
melitensis,  307 

of  sputum  septicemia  (see  Micro- 
coccus  lanceolatus) 
pneumonias    crouposae,    249    (see 
Micrococcus  lanceolatus) 

pyogenes  tenuis  (see  Strepococcus 

pyogenes) 

tetragenus,  in  suppuration,  284 
morphology,  cultural   characters, 

pathogenesis,  300 
ureae,  268 

Micromillimeter,  21 
Micron,  /*,  21 
Microphages,  212 
Microscope,  18 
Microscopic  examination  of  bacteria, 

21 

Microtome,  39 
Miliary  tubercle,  358 
tuberculosis,  358 
effect  of  centrifugal  izing,  154 
of    temperature    on    bacterial 

growth  in,  152 

Milk  as  a  culture-medium,  72 
bacteria  of,  149 
fermentation  in,  152 
germicidal  properties  of,  155 
human  pus  cocci  in,  152 
hygienic  production  of,  158 
lactic  acid  in,  152 


INDEX. 


Milk,  methods  of  determining  number 

of  bacteria  in,  149 
number  of  bacteria  in,  149 
of  lime,  256 

pasteurization,  effect  of,  on  patho- 
genic bacteria  in,  154 

definition  of,  60 
pathogenic  bacteria  in,  151 
poisoning,  153 
pus  cocci  in,  152 
recommendations     of     American 

Public  Health  Association  for 

the   examination  of,  149 
samples  of,  97 

scarlet  fever  conveyed  by,  152 
sources  of  contamination  of,  151 
sterilization    in    infant     feeding, 

*53 

tubercle  bacilli  in,  151,  153 
Miller's  spirillum,  326 
Milzbrand  (see  Anthrax) 
Mixed  infection,  183 
Mixtures  of  toxin  and  antitoxin,  223 
Modes  of  enrty  into  the  body,  172 
Moisture    in    relation    to    growth    of 

bacteria,  126 

Morphology  of  bacteria,  177 
Mosquitoes   as   carriers   of   infectious 

disease:  malaria,  177,  434 
yellow  fever,  440 
destruction  of  (with  coal  oil),  258 

(with  sulphur  dioxide),  255 
Motility  of  bacteria,  124 
Moulds,    aspergillus,  mucor,    oidium, 

penicillium,  278 
cultivation,  76 
description  of,  278 
for  class  work,  115 
in  air,  137 
Mouth,  B.  Friedlander  in,  315 

characteristic  bacteria  in  normal, 

161 

leptothrix  in,  277 
M.  lanceolatus  in,  301,  305 
spirilla  in,.  274 
Movement,  Brownian,  24 
Mucor  mucedo,  279 
Mucous  membranes,  bacteria  of,  160, 

161 

Multiplication  of  bacteria,  122 
Mumps,  microorganism  of,  not  known, 

170 

Mustard  as  a  deodorizer,  257 
Mycelium,  280 
Mycetoma,  369 


Nasal   cavity,   bacteria  of,   normally, 

161 

B.  rhino  scleroma,  316 
Natural  immunity,  199 
Necrosis  caused  by  toxin,  189 
Neisser's  gonococcus  (see  Micrococcus 

gonorrhceae 

stain  for  diphtheria  bacilli,  338 
Neisser-Wechsberg  phenomenon,  233 
Neutral  red  in  culture-media,  71 

in  diagnosis  of  B.coli,  372,  386,  387 
Neutralization  of  culture-media,  65,  66 
Nitrate  of  silver  as  a  germicide,  250 
as  a  stain  for  S.  obermeieri,  405 
Nitrifying  bacteria  in  soil,  131 

nutrition  of,  126 
Nitrites,    conversion    of,    into    nitric 

acid,  131 
Nitrogen  fixation  by  bacteria,  136 

liberation  by  bacteria,  132 
Nitroso-indol  reaction,  132 
Noma,  277 
Non-pathogenic  bacteria,  classification 

of,  267 

Normal  solutions,  66 
Nose-piece  to  microscope,  18 
Novy's  method  for  anaerobes,  32 
Number  of  bacteria  in  feces,  165 
milk,  151 
soil,  135 
water,  139 

species   of    non-pathogenic   bac- 
teria, 267 

Nutrient  agar-agar,  69 
bouillon,  64 
gelatin,  68 
Nutrition  of  bacteria,  126 

Obermeier's  spirillum,  404 

Objectives,  18 

Ocular,  18 

Odors  developed  by  bacteria,  132 

from  water,  139 
Oese,  22 

Oidium  lactis,  279 
Oil,  aniline,  for  Gram's  method,  30 
for  Weigert's  method,  42 

cedar-wood,  20 

immersion  objective,  19 

kerosene,  258 

Oils,  essential,  as  germicides,  257 
Opsonin,  216 
Osteomyelitis,  S.  pyogenes  in,  293 

B.  typhosus  in,  379 
Otomycosis,  281 


INDEX. 


459 


Ovum,  bacteria  conveyed  in,  172 
Oxalic  acid  (see  Acid,  oxalic) 
Oxygen,  relation  of  bacteria  to,  127 
Ox  serum  in  relation  to  cytolysis,  235 
Oysters,   typhoid  fever  conveyed  by, 

158 
Ozena     bacillus    (probably    identical 

with  B.  pneumonia?),  315 
Ozone  in  purifying  water,  142 

Paracolon  bacillus,  384 

Paraffin  imbedding,  38 

Paraform  or  paraformaldehyde,  252 

Paraplague  bacillus,  325 

Parasite,  definition,  120 

Paratyphoid  bacillus,  384 

Parietti's  method  for  examination  of 

water,  147 
Park,  W.  H.,  method  for  cultivating 

anaerobes,  93 
Passive  immunity,  211 
Pasteur,  hydrophobia  virus,  206 

inoculation  of  attenuated  cultures, 

16 
microorganisms,     the     cause     of 

fermentation,  13 
Pasteur-Chamberland  filter,  63 
Pasteurization,  definition,  60 

effect  on  B.  tuberculosis  in  milk, 

153 

Pathogenic  bacteria,  definition,  282 
Pear-blight,  6,  168 
Penicillium  glaucum,  278 
Peptone,  as  product  of  bacterial  growth, 

129 

Dunham's  solution,  73 
enriching  fluid,  399 
in  nutrient  bouillon,  64 
Peptonizing  ferments  formed  by  bac- 
teria, 129 
Pericarditis,    micrococcus   lanceolatus 

in,  306 
staphylococcus    pyogenes    aureus 

in,  288,  294 

streptococcus  pyogenes  in,  296 
Periositis,  due  to  B.  typhi,  379 
Peritonitis,  due  to  B.  coli,  288 

staphylococcus  pyogenes  aureus, 

294 

streptococcus  pyogenes,  296 
B.  pyocyaneus,  318 
B.  proteus,  319 
colon  group,  384 
Perlsucht,  289  ' 
Permanganate  of  potassium,  206,  211 


Peroxide  of  hydrogen,  205 
Petri  dishes,  96 

Petroleum  for  destroying  insects,  258 
Pfeiffer's  capsule  bacillus,  315 
Pfeiffer's  phenomenon)  228,  394 
Phagocytosis,    as    means   of    defense 
against  infection,  282 

definition,  212 

melaniferous,  437 
Phenol  (see  Acid,  carbolic) 
Phenolphthalein  in  titration,  66 
Phosphorescence    of    bacteria,     128, 

271 

Physiology  of  bacteria,  117 
Picric  acid  (see  Acid,  picric) 
Piorkowski's  culture-medium,  374 
Piroplasma,  177,  440 
Placenta,  bacteria  transmitted  through, 

172 
Plague,     bubonic,    bacillus    of    (see 

Bacillus  pestis  bubonicae) 
Plants,  diseases  of,  6,  168 
Plasmodium  of  malaria,  morphology, 
cycle  of  development,  432 

preparation  of  blood-films,  108 

staining  of,  45 
Plasmolysis,  121 
Plate-cultures,  95 

Platinum  wire,  rules  for  use,  22,  78 
Pleuritis,     streptococcus     pyogenes 
aureus  in,  288,  294 

streptococcus  pyogenes  in,  296 

micrococcus  lanceolatus  in,  306 
Pleuro-pneumonia  of  cattle,  150 

invisible  microbes,  171 
Plugs,  cotton,  for  tubes,  76 

for  anaerobic  cultures,  91 
Pneumococcus  of  Frankel  (see  Micro- 
coccus  lanceolatus) 

Pneumonia,    broncho-,    due   to   acid- 
proof  bacteria,  370 

to  B.  pneumonias,  315 

to  B.  pycyaneus,  318 

to  B.  typhosus,  379 

to  pus  cocci,  288 

croupous    (see    Croupus    pneu- 

moniae) 

Pneumonomycosis,  281 
Poisoning  by  food,  milk,  ice  cream, 
cheese,  153 

oysters,  fish,  meat,  159 
Porcelain  filter,  62 

Post-mortems,     disinfection     at,     on 
animals,  95 

on  human  beings,  259 


460 


INDEX. 


Post-office  rules  for  mailing  specimens 

of  bacteria,  no 
Potassium  permanganate,  257 
Potato  as  a  culture-medium,  71 
growth  of  B.  typhosus  on,  372 
bacillus  (see  Bacillus  mesentericus 

vulgatus) 
Precipitins  for  albumen,  196 

for  bacteria,  197 
Predisposition  to  infection,  155 

age,  altitude,  climate,  individual, 

179 

amount     of    infectious  material, 
number  of  bacteria,   virulence 
of 'bacteria,  182 
bodily    conditions,   general,    178. 

181 

local,  285 
of  different  organs  and  tissues, 

racial,  180 

Pressure,  effect  of,  on  bacteria,  247 
Prevention    and    cure    of    bacterial 

diseases,  16 

Products,  bacterial:    ammonium    car- 
bonate, fatty  acids,  indol,  leu- 
cin,      phenol,      skatol,     sugar, 
toxins,  tyrosin,  129 
pigment,  128 
ptomaines,  159,  161 
tyrotoxicon,  153 

Propionic  acid  (see  Acid,  proprionic) 
Protargol,  250 
Protective    inoculation    for    anthrax, 

201,  335 

Asiatic  cholera,  394 
black-leg  of  cattle,  202 
bubonic  plague,  202,  323 
erysipelas  of  swine,  202 
fowl-cholera,  202 
hydrophobia  or  rabies,  206 
rinderpest,  201 
small-pox,'  10,  200 
tuberculosis,  362 
typhoid  fever,  381 

Proteus  bacillus  (see  Bacillus  proteus) 
Protoplasm,  appearance  of  bacterial, 

121 

Protoxoid,  224 
Protozoa,    pathogenic,    in    dysentery, 

malaria,  17 
Texas  fever,  177 
trypanosomes,  408 
amebae,  410 

Pseudo-diphtheria  bacillus,  343 
Pseudo-gonococcus,  311 


Pseudo-membranous    inflammations 
due  to  B.  diphtheriae,  342,  345 
B.  dysenteriae,  307 
M.  lanceolatus,  307 
streptococcus  pyogenes,  296 
Pseudo-pneumococcus,  307 
Pseudo-tuberculosis,  362 
Ptomaine  poisoning,  159 
Ptomaines,  186,  187 
Puerperal  fever,  n 

B.  diphtheria  in,  345 
S.    pyogenes  in,  296 
Pure  cultures,  advantages  of,  shown 

by  Koch,  16 

methods  of  obtaining,  by  animal 
inoculation  by  dilution,  by  heat- 
ing, 94 

by  plating,  104 
Purification  of  water  by  chemicals,  by 

mechanical  filtration,  141 
by  sand  filtration,  140 
Pus,  blue,  318 
cells,  282 

collection    of   specimens   for   ex- 
amination, 107,  109 
formation,  283 
green,  318 

Putrefaction,  13,  133 
Pyemia,  291 
Pyocyanase,  190 
Pyocyanin,  317 
Pyogenic  bacteria,  as  causes  of  various 

diseases,  288 

as  secondary  invaders,  287 
in    inflammation   of   middle-ear, 

289 

in  pneumonia,  289 
in  suppuration,  284 
Pyoktanin,  250 
Pyosalpinx,  314 

Pyrogallic   acid  for  cultivating  anae- 
robes, 91 
Pyroxylin,  38 

Quarantine,  early  used  by  Italians,  9 

Rabies,  caustive  agent  unknown,  170 

diagnosis  of,  208 

Pasteur  treatment  for,  virus  fixe, 

206 
Racial  immunity,  199 

predisposition  to  infection,  179 
Rats,  acid-proof  bacilli  of,  362 

relation  to  bubonic  plague,  322 
Rauschbrand,  vaccine  for,  202 


INDEX. 


461 


Ray-fungus  of  actinomycosis,  in  pus 

formation,  284 
morphology,    cultural     character 

and  pathogenesis,  366 
Reactions  of  culture-media,  correction 

of,  68 

methods  of  determining,  65 
.optimum,  67,  127 
Reactivated  blood-serum,  195,  222 
Receptor,  antitoxin,  definition,  218 
first  order,  221 
second  order,  221 
third  order,  223 
Recovery     from     infectious     disease, 

agents  concerned  in,  184 
influence  on  immunity,  199,  212 
Regulators,  gas,  Reichart's,  84 
Roux,  85 

electric,  Roger's,  87 
Relapsing  fever,  spirillum  (see  Spiril- 
lum obermeieri) 

Rennet-like  bacterial,  ferments,  129 
Rheumatism,    microorganism    doubt- 
ful, 170 
M.   rheumaticus,   pyogenic  cocci 

in,  290 

Rhinosderoma,  bacillus,  316 
Ricin,  analogy  with   bacterial  toxins, 

189 

Ripening  of  cream,  151 
Robin,  analogy  with   bacterial  toxins, 

189 
production    of     antitoxin     with, 

209 

Roll-tubes  of  Esmarch,  99 
Rooms,  disinfection  with  formalin,  253 

with  sulphur  dioxid,  254 
Rontgen-rays,    influence   of,    on   bac- 
teria, 247 

Root-tubercle  organisms,  on  legumin- 
ous plants,  organisms  of,  136 
Rosolic  acid,  (see  Acid,  rosolic) 
Rouget,  202 

Rubber  caps  for  culture-tubes,  86 
for  use  at  autopsies,  no 
at  surgical  operations,  265 
gloves,  212 
stoppers  for  culture-tubes,  86 

Sabouraud's  culture-medium  for  fungi, 

76 

Saccharomyces  cerevisiae,  278 
Saccharose,  agar,  71 

bouillon,  67 
Salt-agar,  264 


Sanarelli's    bacillus   of    yellow   fever 

(see  Bacillus  icteroides) 
Sand  filtration  of  water,  140 
Sapremia,  184 
Saprophyte,  definition,  167 
Sarcina,  definition  of,  119 
pulmonum,  268 
ventriculi,  268 
in  healthy  stomachs,  164 
morphology,   cultural  characters, 

268 
Sarcoma,  toxins  of  streptococcus  for, 

299 

complicated  with  diphtheriae,  345 
microorganism  of,  not  discovered, 

170 

streptococcus  in,  297 
Scarlet  fever,  170 
Schizomycetes,  definition,  4 
Schultz's     method     for     neutralizing 

culture-media,  65 
Schweinerothlauf,  vaccine  for,  202 
Scrofula,  358 
Sealing    culture-tubes    for   anaerobic 

cultures,  91 
to  prevent  drying  out  of  culture 

medium,  86 

Secondary  infection,  183 
definition  of,  183 
due  to  S.  pyogenes,  287 
Section-cutting,  40 
Sections,  stained  with  carmine,  45 
staining  bacteria  in,  41 
Gram's  method,  42 
hematoxylin,  44 
tubercle  bacilli,  43 
Weigert  method,  42 
Sedgwick's  test  for  germicides,  244 
Sedgwick-Tucker  aerobioscope,  138 
Self -purification  of  water,  139 
Semen,  transmission  bacteria  by,  172 
Semmelweis,   demonstration  of  infec- 
tious nature  of  puerperal  fever, 
ii 

Separator  for  milk,  influence  on  num- 
ber of  bacteria,  154 
Septicemia,  184 
Serum  (see  Blood-serum) 

test  for  typhoid  fever,  value  of, 

J93 

method  employed  in,  376 
Shiga's    bacillus     of     dysentery    (see 

Bacillus  dysenteric) 
Side-chain  theory  of  immunity,  218 
Silk  threads  in  testing  germicides,  243 


462 


INDEX. 


Silver  nitrate,  250 

wire  in  surgery,  266 
Size  of  bacteria,  4,  121 
Skatol,  produced  by  bacteria,  129 
Skin,  bacteria  of,  160 

disinfection,  161;  265 
Sleeping  sickness,  170 
Slides,  forceps  for,  27 

glass,  27 
Small-pox,  bacteria  found  in,  437 

cytoryctus  variolas,  422 

inoculation  of,  10 

vaccination  for,  200 
Smear-culture,  80 

preparations,  26 
Smegma  bacilli,  33,  163 
Snake-venom,  189 
Sodium  hydroxide,  for  neutralization 

of  culture  media,  64,  65,  66 
Soft  chancre,  bacillus  of   (see  Bacillus 

of  soft  chancre) 
Soil,  bacteria  of,  135,  176 
Solutions,  normal,  66 
Species  of  bacteria,  117 
Spirilla  in  the  mouth,  161 

for  class  demonstration,  114 

in  water,  403 

S.  dentium,  274 

S.  milleri,  326 

S.  plicatile,  S.  rugula,  S.  volutans, 
274 

S.  sputigenum,  274 

vibrio    aquatiles,    vibrio    Schuyl- 

kiliensis,  403 
Spirillum,  definition,  4,  118,  120 

choleras  Asiatici,  accidental  infec- 
tion  of   laboratory  workers, 

395 
description     of,      morphology, 

cultural    characters,    patho- 

genesis,  317 

detection  of,  in  feces,  322 
diagnosis  of,  322 
disseminated     through    water, 

145 

in  milk,  152 

killed  by  the  hydrochloric  acid 
of  the  gastric  juice,  164,  393 

pathogenic  properties  for  exper- 
iment animals,  393 

portals  of  entry  into  the  body, 

395 
reported  detection  of,  in  water, 

145 
vaccine  for,  394 


Spirillum,  dentiumj  274 

of  Deneke,  402 

of  Finkler  and  Prior,  401 

of  Metchnikoff,  400 

of  Miller,  402 

of  Obermeier,  404 

of  Vincent,  275 

plicatile,  274 

relapsing  fever,  404 

rubrum,  274 

rugula,  274 

sputigenum,  274 

tyrogenum,  402 

undula,  274 

volutans.,  274 
Spirochaeta,  definition,  120 

dentium,  274 

Obermeieri,  404 

of  syphilis,  405 

pallida,  405 

plicatile,  274 

refringens,  406 
Splenic  fever  (see  Anthrax) 

puncture  in  typhoid  fever,  378 
Spontaneous  generation,  3,  13 
Spores  .arresting  and  resisting  forms  of 
bacteria,  3 

discovery  of,  by  Cohn,  14 

reproduction  of  bacteria  by,  122 

arthro,  123 

compared    with   vegetative   cells, 

122 

definition  of,  3 

discovery  of,  14 

endogenous,  122 

of  moulds,  280 

of  the  malarial  parasite,  416,  418 

resistance    to    heat     and     other 
germicides,  123 

staining,  48 

Sporotricha  or  sporothrix,  232 
Sputum,  disinfection  of,  34,  259 

method  of  making  preparations 
of,  34,  1 08 

rules  for  collection  of,  34,  107 

staining  of,  34 
Stab-culture,  78 
Staining  anilin-oil,  water  solution,  30 

bacteria  in  tissues,  37,  41 

blood.  45 

Hasting's  method,  46 
Nocht's,  Goldboron's,  Wright's, 

47 
capsules,  49 

Hiss'  method,  Welch's,  49 


INDEX. 


463 


Staining,  cover-glass  preparation,  29 
diphtheria  bacillus,  338 
flagella,  50 

Gabbett's  solution,  35 
gonococcus,  311 
Gram's  method,  31 

bacteria  which  stain  by,  32 
Gram-Weigert  method,  41 
malarial  parasite,  45 
neuclei  of  tissues,  44 
sections,  41 
spores,  48 

tubercle  bacillus  in  milk,  33 
in  sputum,  35,  108 
in  tissue,  41 
Stalactite  growth  of  plague    bacillus, 

321 
Staphylococcus    cereus    albus,  cereus 

flavus,  284 
definition,  118 

epidermidis  albus  in  milk,  152 
in  normal  skin,  161,  263 
in  suppuration,  284 
morphology,  cultural  character, 

pathogenesis,  294 
pyogenes  albus  in  cystitis,  290 
in  milk,  152 
in  normal  skin,  161 
in  secondary  infection,  287 
in.  suppuration,  284 
in  wounds,  286 
morphology,  cultural  charac- 
ters, pathogenesis,  294 
aureus  in  cystitis,  290 
in  milk,  152 
in  normal  skin,  161 
in  secondary  infection,  287 
in  suppuration,  284 
in  wounds,  286 
morphology,    cultural    char- 
acters, pathogenesis,  291 
citreus,  284 
in  cystitis,  290 
in  milk,  152 
in  normal  skin,  161 
in  secondary  infection,  287 
in  suppuration,    284 
in  wounds,  286 
Steam  sterilization,  55 

(in  contagious  cases),  260 
(on  animals),  104 
Stegomyia,  154,  336 
Sterilization,  55 

at  autopsies  (on  animals),  104 
(in  contagious  diseases),  260 


Sterilization,   by   boiling  for  various 
purposes,  55 
for  drinking  water,  142 
for  surgical  instruments,  266 

by  nitration,  62 

by  pasteurization,  60 

by  steam,  55 

by  the  autoclave,  60 

by  the  naked  flame,  53 

fractional,  55 

hot-air,  53 

intermittent,  55 

methods  of  testing  efficacy  of,  115 

of  blood-serum,  59,  74 

of  brushes  used  in  surgery,  266 

of  culture-media,  blood-serum,  74 
by  filtration,  64 
by  live  steam,  55 
by  pasteurization,  60 
in  autoclave,  60 

of  cultures,  96,  116 

of  dressings,  265 

of  glassware,  53 

of  gloves,  rubber,  265 

of  hands,  264 

of  hypodermic  solutions,  266 

of  ligatures,  265 

of  milk  in  infant  feeding,  153 

of  platinum  needle,  79 

of  steam,  51,  55 

of  surgical  instruments,  266 

of  test-tubes,  76 

of  water,  142 
Sterilizer,  Arnold,  56 

hot-air,  53 

Koch,  55 

Massachusetts  Board  of  Health, 58 

steam,  55 
Sternberg's  bulbs,  no 

determination  thermal  death-point 

of  bacteria,  125 
Stewart's  forceps,  26 
Stick-culture  (see  Stab-culture) 
Stitch- abscesses,  266 
Stomach,  bacteria  of,  164 
Stools,  disinfection,  259 
Straus's     method    for     diagnosis     of 

glanders,  365 
Streak  cultures,  80 
Streptococcus  brevis,  295 

definition,  118 

injection  of  cultures  of,  for  cure  of 
sarcoma,  299 

lanceolatus      (see      Micrococcus 
lanceolatus) 


464 


INDEX. 


Streptococcus,  longus,  296 
of  erysipelas,  300 
polyvalent  serum  for,  298 
pyogenes  (see  also  Suppuration). 

294 

en  do  toxin  of,  190 
in  suppuration,  284 
morphology,  cultural  characters, 

pathogenesis,  294 
secondary  infection  with,  287 
serum,  247 

Streptothrix,  228,  276,  369 
actinomyces,  366 
cuniculi,  277 
Stropping  knives,  40 
Substance  sensibilisatrice,  226 
Sugar-free  bouillon,  67 
Sugars  in  culture-media,  67,  71 
Sulphur  dioxide,   use  in  disinfection, 

209,  254 

Sunlight,  influence  on  bacteria,  247 
Suppuration,  14,  282 
Surgical  disinfection,  262 
Surra,  409 
Swarming     islands     of     B.     proteus, 

263 

Swine  erysipelas,  vaccine  for,  202 
Symptomatic  anthrax,  202 
Symptomatic   anthrax,  production  of 

immunity  in,  202 
Syntoxoid,  223 
Syntoxonoid,  224 
Syphilis,  various  organisms  in,  171 

spirochaetae  in,  405 
Systematic  study  of  species  of  bacteria, 


Teaching     bacteriology,     suggestions 

for,  113 

Teeth,  bacteria  of,  162 
Terminal  infections,  183 
Test-tubes  inoculation  of,  78 

kind    best   adapted    to  bacterio- 
logical work,  76 

manner  of    filling  with    culture- 
media,  77 
of  holding,  78 
plugs  for,  76 
sealing  of,  for  anaerobic  cultures, 

91 

to  prevent   drying   of  the   me- 
dium, 86 

sterilization  of,  77 

Tetanus  antitoxin,  principles  involved 
in  the  production  of,  209,  331 


Tetanus  bacillus,  84,  122,  269 
in  soil,  135 

isolation  of  cultures  by  heat,  94 
morphology,    cultural    charac- 
ters, pathogenesis,  327 
toxin,  162 

different  in  action  from  poisons, 

188 

agents  distructive  to,  330 
extracellular  character,  187 
Tetrad,  definition,  119 
Texas  fever,  177 
Thermal     death-point     of     bacteria, 

determination,  125 
Thermophilic  bacteria,  125 
Thermostat  (see  Gas-regulator) 
Thiothrix,  277 
Thrush,  280 
Thymol,  107 

Tinea  favosa,  trichophytina,  281 
Tissues,  fixation  and  hardening,  37 

staining  bacteria  in,  37,  41 
Titration  of  culture-media,  65 
Toxemia,  184 
Toxin,  definition,  187 
endo-,  200 
of    diphtheria    and    of    tetanus, 

extracellular  nature,  188 
of   diphtheria   mixed   with   anti- 
toxin, 223 
potency  of  189, 
resemblance  to  ricine,  209 
Toxins  as  end-products  of  bacterial 

growth,  1 29 
extracellular,    intracellular,     187, 

200 

in  production  of  antitoxin,  209 
necrosis  produced  by,  189 
potency  of,  189 
spectra  of,  224 
Toxoid,  225 
Toxon,  225 
Toxonoid,  225 
Toxophore     group     of     the    ambo- 

ceptor,  219 

Trichophyton,  cultivation,  76 
Trypanasoma      brucei,      dimorphon, 
equinum,     equiperdum,    evansi, 

gambiensis,  theileri,  410 
of  nagana,  410 
of  surra,  410 
Trypanosomes,  407 
cultivation  of,  408 
description  of,  408 
Tsetse-fly  disease,  in  surra,  77,  409 


INDEX. 


465 


Tubercle  bacillus  (see  Bacillus  tuber- 
culosis) gray,  miliary,  yellow,  358 
structure,  356 

Tuberculin,  method  of  preparation,36o 
R,  361 

Tuberculosis,  356 

acute  miliary,  358 

bovine,  avenues  of  infections  in, 

359 
cross-infection  of    human  beings 

and  of  cattle,  355 
diagnosis   by   agglutination    test, 

360 

by  cover-glass  preparations,  33 
by  culture,  350 
by  tuberculin  test,  36 
frequency,  151,  356 
immunity,  from,  362 
infection  from  milk  of,  151 
infection  of  infants,  151 
infection  of  meat  of  cattle  suffer- 
ing from,  152 
of  birds,  362 
organs  affected  by,  358 
pseudo-,  362 

spread  of,  in  the  body,  358 
tuberculin  diagnosis  of,  361 
Transmission  through  placenta,  173 
Typhoid    fever   bacillus     (see   Typhi 

abdominalis) 
Typhus  fever,  170 
Tyrosin,  produced  by  bacteria,  129 
Tyrotoxicon,  153 

Ult ramie roscopic  organisms,  171 

Unit,  immunity,  348 

Urea,  decomposition  by  bacteria,  129 

Urethra,  bacteria,  164 

Urethritis,  gonorrheal,  312 

Urinary  bladder,  bacteria  of  (see  also 
Cystitis),  160 

Urine,  disinfection,  259 

preservation  of  samples  of,  107 
serum-agar,     for    cultivation    of 

gonococcus,  313 
typhoid  bacilli  in,  378 

Uterus,  bacteria  of,  160 

Vaccination,  n,  171 

tetanus  following,  329 
Vaccines,  bacterial,  16,  201 

black-leg,  202 

cholera,  394 

COW-pOX,    2OO 

hydrophobia,  203 
3° 


Vaccines,  plague,  202 
streptococcus,  298 
swine  erysipelas,  202 
Vaccinia,    bacteria    in,    protozoa    in, 

421 

Vagina,  bacteria  of,  162 
Vaginitis,  gonorrheal,  314 
Van  Ermengem's  method  for  staining 

flagella,  51 

Vegetative  forms  of  bacteria,  122 
Venom  of  snakes,  189 
Vibro  aquatilis,  403 
Berolinensis,  402 
definition,  120 
Metchnikovii,  400 
proteus,  401 
rugula,  274 
Schuylkiliensis,  403 
Vibrion   septique    (see   Bacillus   oede- 

matis  maligni) 
Villemin,  first  to  produce  tuberculosis 

by  inoculating  animals,  12 
Vincent,  bacillus  of,  275 
Vinegar,  bacteria  in,  5 
Violet,  gentian-,  aniline-oil,  30 

aqueous,  23 
methyl-,  251 
Virulence  of  bacteria,  effect  of  artificial 

cultivation  on,  82 
influence  of  moisture  on,  126 
variability  due  to  sundry  causes, 
182 

Warmth,  relation  to  growth  of  bacteria, 

I25 

Water,  B.  coli  communis  in,  146 
B.  typhosus  in,  145 
bacteria  of,  139 

conveyed  by,  139 

Water,    collection  of  samples  for  ex- 
amination, 142 
detection  of  pathogenic  bacteria 

in,  145 
estimation    of    the     number    of 

bacteria  in,  143 
filtration,  63,  140 
ground-,  139 
infections  carried  by,  139 
pathogenic  bacteria  in,  139,  145 
purification  by  ozone,  142 
recommendations     of    American 

Public  Health  Association,  147 
self-purification,  139 
spirilla  in,  114,  274,  403 
S.  cholerae  Asiatici  in,  145 


466 


INDEX. 


Water,  sterilization  of,  142 
storage  of,  140 

transmission    of    typhoid    fever, 
of  cholera,  of  dysentery  by,  176 
transportation    of,    for    examina- 
tion, 142 

typhoid  fever  bacilli  in,  145 
well,  contamination  of,  139 
Watery  solutions  of  aniline  dyes,  28 
Weigert's  stain  for  fibrin  and  bacteria, 

42 

Welch's  stain  for  capsules,  49 
Whooping-cough,  171 
Widal's  serum-test  for  typhoid  fever, 

376 
Wire  baskets,  77 

platinum,  22 

silver,  266 

Wolffhiigel  plate,  144 
Wool-sorter's  disease,  137,  176,  334 
Wounds,  infected,  265,  181,  286 
Wright's  stain  for  blood,  47 

method  for  anaerobes,  91 
Wurtz's  culture-medium,  371 
Wurzelbacillus  (see  Bacillus  mycoides) 


Xerosis  bacillus,  343 
X-rays,  247 
Xylol,  38,  41 

Yeasts,  description  of,  273 
for  class  work,  115 
in  air,  137 
Yellow  fever,  bacteria  not  the  cause 

of,  424 

conveyed  by  mosquito,  440 
destruction  of  mosquito  of,  by 

sulphur  dioxide,  255 
by  petroleum,  258 
filterable  virus,  172 
microorganism  unknown,  170 
tubercle,  358 
Yersin's  serum  for  plague,  324 

Ziehl's  carbol-fuchsin,  34 

Zinc  chloride,  sulphate,  258 

Zooglcea,  122 

Zomotoxic  group  of  the  amboceptor, 

222 

Zymophore  group  of  the  amboceptor, 


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